Genetically modified rat derived from rat embryonic stem cell

ABSTRACT

The present invention provides a rat embryonic stem cell characterized by having the following properties of (a) expressing Oct3/4 gene and Nanog gene, (b) positive for alkaline phosphatase activity, (c) having an embryoid body forming ability, (d) expressing SSEA (Stage-Specific Embryonic Antigen)-1 and SSEA-4, (e) having the same number of chromosomes as does a normal rat cell, (f) capable of being subcultured and holding the undifferentiated state, (g) having in vitro pluripotency, (h) having a potential to differentiate for cells of three embryonic germ lineages, (i) having teratoma formation ability, and (j) having an ability to produce a chimeric rat, a method of establishing the aforementioned rat embryonic stem cell and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of copending U.S. patentapplication Ser. No. 14/132,969, filed on Dec. 18, 2013, which is adivisional of U.S. patent application Ser. No. 13/370,795, filed on Feb.10, 2012, now issued as U.S. Pat. No. 8,628,957, which is a divisionalof U.S. patent application Ser. No. 10/591,407, filed on Dec. 8, 2006,now issued as U.S. Pat. No. 8,137,966, which is the U.S. national phaseof International Patent Application No. PCT/JP2005/003841, filed on Mar.1, 2005, which claims the benefit of Japanese Patent Application No.2004-310465, filed on Oct. 26, 2004, and Japanese Patent Application No.2004-061300, filed on Mar. 4, 2004, all of which are incorporated byreference in their entireties herein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 6,261 bytes ASCII (Text) file named“728976SequenceListing.txt,” created Jul. 7, 2017.

TECHNICAL FIELD

The present invention relates to rat embryonic stem cells (hereinafterto be referred to as ES cells). More particularly, the present inventionrelates to rat ES cells, production methods of rat ES cells, subculturemethods of rat ES cells, screening methods for a differentiationinduction-related substance using rat ES cells, use of theaforementioned rat ES cells for the production of a genetically modifiedrat and the like.

BACKGROUND ART

ES cells are cells of cell lines established from an inner cell mass ofblastocysts, and autonomously replicable in the presence of a leukemiainhibitory factor (LIF). ES cells can be differentiated into any type ofcells (neuron, myocyte, vascular endothelial cell, erythrocyte,leukocyte, platelet, bone, cartilage, kidney, intestine, liver,pancreas, lung and the like) by changing culture conditions. Genomes ofmany kinds of animals have been decoded as the results of genomeprojects, and the information of their homologies with humans has beenaccumulated. The destruction of a particular gene in the stage of EScells based on these information may elucidate a role (function) of thegene in cell differentiation, or growth of individual, homeostasis orthe like. Specifically, a chimeric animal can be produced by injectingES cells, in which a particular gene has been destructed, into normalhost blastocysts to be mixed with the cells of the host embryos andreturning the mixture to a uterus, and an animal, in which theparticular gene has been destructed (knockout animal), can be producedby crossing the obtained chimeric animals. In addition, the effects of acompound on genes in various cells (organs) can be evaluated by applyingthe compound to ES cells. On the other hand, a use of normal cellsobtained by differentiating ES cells enables cell therapy andregenerative medicine. As mentioned above, ES cells can be widelyapplied to studies in physiology, pharmacology, regenerative medicineand the like. However, in experimental animals, ES cells derived fromonly mouse (Evans M. J. et al., Nature 1981, 292: 154-156), rhesusmonkey (Thomson J. A. et al., Proc. Natl. Acad. Sci. USA 1995, 92:7844-7848), marmoset (Thomson J. A., et al., Cur. Top. Dev. Biol. 1998,38: 133-165) and crab-eating monkey (Suemori H, et al., Methods Enzymol.2003, 365: 419-429) have been established, and a rat ES cell has notbeen established yet.

Rat is a mammal having a size suitable for experiments (about 10 timesthe size of mouse), and it is advantageous in that (1) a drug can beeasily administered to a minute blood vessel, (2) surgical andtransplant experiments can be performed and (3) a large amount of tissuecan be obtained. Many human disease model rats have been developed anddiscovered. Rat is one of the most useful experimental animals, which iswidely utilized in various fields including medicine. For the past 100years, rat has been utilized for the studies in functions and the likeas a model for cancer, cerebral nerve system, transplantation and humanmultifactorial disorder, and a vast amount of functional researchresources has been accumulated. Particularly, the analysis of brainatlas is ongoing and abundant information of behavioral study inpsychophysiology is available. As regards gene analysis, which is theweakest point as compared to mouse, too, since the analysis of ratgenome is advancing, comparison with human at the gene level has beenenabled, and rat has been drawing attention as an excellent experimentalanimal leading the post-genome. In fact, the Bioresource AreaSubcommitte, the Life Science Subcommittee, the Subdivision on R&Dplanning and Evaluation, the Council for Science and Technology held inMarch, 2002 admitted that rat was one of the bioresources that should beenriched in Japan in the future, and that establishment of ES cells forthe purpose of production of knockout rats was necessary.

Despite such utilities of rat and enrichment of genetic informationtherearound and the like, it has been extremely difficult to establishrat ES cells essential for the production of a genetically modified rat.For example, Iannaccone, P. M. et al. (Developmental Biology 1994, 163:288-292) and the corresponding patent application WO 95/06716 describethat ES cells have been established from the rat strain PVG and chimericrats have been produced. Thereafter, however, Brenin, D. et al.(Developmental Biology 1997, 185: 124-125) describe that the chimericrats produced in Developmental Biology 1994, 163: 288-292 were producedby the contamination of mouse ES cells. Namely, it has been shown thatthey did not succeed in the establishment of rat ES cells and theproduction of chimeric rat. There are some other cases where theestablishment of rat ES cell was attempted (see WO 99/27076 and JP2002-176973 A), but all of them have not succeeded in the establishment.

As mentioned above, plural research groups have unsuccessfully tried toestablish rat ES cell lines. In the INTERNATIONAL CONGRESS ON STEM CELLSheld in Keystone in April 2004 in the United States, it was recognizedthat there had been no report on the establishment of rat ES cells allover the world. A key to the successful establishment of rat ES cells isthe setting of culture conditions for the production of ES cells. Whilethe conventional attempts have been made based on the establishment ofand culture conditions for mouse ES cells, rat ES cells have not beenestablished as yet. From these backgrounds, it is considered thatingenuity in the culture conditions is essential for the production ofrat ES cells.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide rat ES cells notavailable heretofore. It is also an object of the present invention toprovide a method of establishing and producing rat ES cells, a method ofsubculturing rat ES cells, a method of screening for a differentiationinduction-related substance using rat ES cells, and a use of theaforementioned rat ES cells for the production of a genetically modifiedrat and the like.

The present inventors tried to establish rat ES cells from blastocystsderived from various rat strains and determined culture conditions andpassage conditions, which are completely different from those for mouseES cells, thereby successfully establishing rat ES cell lines.Specifically, the present inventors first succeeded in establishing andsupplying rat ES cells, which are stable and meet all of therequirements of ES cells, by the use of a culture medium substantiallyfree of serum, and by trying various measures such as attemptingestablishment by mechanical means for isolation of inner cell mass andpassage of ES cells.

The present invention has been completed based on these findings.

Accordingly, the present invention is listed in the following:

-   (1) a rat embryonic stem cell characterized by having the following    properties (a)-(j):-   (a) expressing Oct3/4 gene and Nanog gene,-   (b) positive for alkaline phosphatase activity,-   (c) having an embryoid body forming ability,-   (d) expressing SSEA (Stage-Specific Embryonic Antigen)-1 and SSEA-4,-   (e) having the same number of chromosomes as does a normal rat cell,-   (f) capable of being subcultured and holding the undifferentiated    state,-   (g) having in vitro pluripotency,-   (h) having a potential to differentiate for cells of three embryonic    germ lineages,-   (i) having teratoma formation ability,-   (j) having an ability to produce a chimeric rat,-   (2) the rat embryonic stem cell of the aforementioned (1), further    having the property:-   (k) differentiating by culture in the presence of 20% serum,-   (3) a rat embryonic stem cell obtained by performing a process    comprising the following steps (A)-(D), under the culture conditions    using a substantially serum free culture medium:-   (A) a step for dissociating an inner cell mass formed by the culture    of rat blastocysts, remaining a state of cell aggregate,-   (B) a step for culturing primary embryonic stem cells resulting from    the culture of the dissociated inner cell mass until it can be    passaged,-   (C) a step for dissociating the primary embryonic stem cells, which    have become capable of being passaged, remaining a state of cell    aggregate, followed by passaging and culturing the same,-   (D) a step for further passaging and culturing the cells to    establish an embryonic stem cell,-   (4) the embryonic stem cell of the aforementioned (3), wherein the    culture medium comprises a serum replacement reagent,-   (5) the embryonic stem cell of the aforementioned (3) or (4),    wherein the step (A) comprises a step for mechanically dissociating    the inner cell mass,-   (6) the embryonic stem cell of any of the aforementioned (3)-(5),    wherein the step (C) comprises a step for mechanically dissociating    the embryonic stem cells,-   (7) the embryonic stem cell of any of the aforementioned (3)-(6),    wherein a culture medium without rat leukemia inhibitory factor    (rLIF) is used in step (A),-   (8) the embryonic stem cell of any of the aforementioned (3)-(7),    wherein an rLIF-containing culture medium is used in steps (B)-(D),-   (9) the embryonic stem cell of any of the aforementioned (3)-(8),    wherein feeder cells are used in the culture,-   (10) the embryonic stem cell of the aforementioned (9), wherein the    feeder cells are embryo-derived normal fibroblasts,-   (11) the embryonic stem cell of any of the aforementioned-   (1)-(10), which is derived from either strain of Wistar Kyoto strain    (WKY), Wistar Hannover GALAS strain (WHG) and Brown Norway strain    (BN),-   (12) a production method of a non-mouse embryonic stem cell which    comprises performing a process comprising the following steps    (A)-(D), under the culture conditions using a substantially serum    free culture medium:-   (A) a step for dissociating an inner cell mass formed by the culture    of rat blastocysts, remaining a state of cell aggregate,-   (B) a step for culturing primary embryonic stem cells resulting from    the culture of the dissociated inner cell mass until it can be    passaged,-   (C) a step for dissociating the primary embryonic stem cells, which    have become capable of being passaged, remaining a state of cell    aggregate, followed by passaging and culturing the same,-   (D) a step for further passaging and culturing the cells to    establish an embryonic stem cell,-   (13) the production method of the aforementioned (12), wherein the    non-mouse embryonic stem cell is a rat embryonic stem cell,-   (14) the production method of the aforementioned (12) or (13),    wherein the culture medium comprises a serum replacement reagent,-   (15) the production method of any of the aforementioned (12)-(14),    wherein the step (A) comprises a step for mechanically dissociating    the inner cell mass,-   (16) the production method of any of the aforementioned (12)-(15),    wherein the step (C) comprises a step for mechanically dissociating    the embryonic stem cells,-   (17) the production method of any of the aforementioned (12)-(16),    wherein an rLIF-free culture medium is used in step (A),-   (18) the production method of any of the aforementioned (12)-(17),    wherein an rLIF-containing culture medium is used in steps (B)-(D),-   (19) the production method of any of the aforementioned (12)-(18),    wherein feeder cells are used in the culture,-   (20) the production method of the aforementioned (19), wherein the    feeder cells are embryo-derived normal fibroblasts,-   (21) the production method of any of the aforementioned (13)-(20),    wherein the rat embryonic stem cell is derived from either strain of    Wistar Kyoto strain (WKY), Wistar Hannover GALAS strain (WHG) and    Brown Norway strain (BN),-   (22) a subculture method of non-mouse embryonic stem cells which    comprises dissociating and passaging the cells, remaining a state of    cell aggregate,-   (23) the subculture method of the aforementioned (22), wherein the    non-mouse embryonic stem cell is a rat embryonic stem cell,-   (24) the subculture method of the aforementioned (22) or (23), which    comprises a step for mechanically dissociating the cells,-   (25) the subculture method of any of the aforementioned (22)-(24),    wherein the cells are cultured using a culture medium substantially    free of serum,-   (26) the subculture method of the aforementioned (25), wherein the    culture medium comprises a serum replacement reagent,-   (27) the subculture method of the aforementioned (25) or (26),    wherein the culture medium comprises rLIF,-   (28) a culture medium for non-mouse embryonic stem cell, which    comprises a serum replacement reagent and rLIF,-   (29) the culture medium of the aforementioned (28), wherein the    non-mouse embryonic stem cell is a rat embryonic stem cell,-   (30) a culture kit for non-mouse embryonic stem cell, which    comprises a serum replacement reagent and rLIF as components,-   (31) the culture kit of aforementioned (30), wherein the non-mouse    embryonic stem cell is a rat embryonic stem cell,-   (32) the culture kit of the aforementioned (31), which further    comprises the rat embryonic stem cell of the aforementioned (1)-(11)    as a component,-   (33) the culture kit of any of the aforementioned (30)-(32), which    further comprises feeder cells,-   (34) the culture kit of the aforementioned (33), wherein the feeder    cells are embryo-derived normal fibroblasts,-   (35) a differentiation induction method of a rat embryonic stem    cell, which comprises stimulating the rat embryonic stem cell of the    aforementioned (1)-(11) with a differentiation inducer,-   (36) the differentiation induction method of the aforementioned    (35), wherein the differentiation inducer is a retinoic acid, growth    factor, glucocorticoid or extracellular substrate,-   (37) a cell obtained by inducing the differentiation of the rat    embryonic stem cell of any of the aforementioned (1)-(11),-   (38) a cDNA library, genomic library or cell extract derived from    the rat embryonic stem cell of any of the aforementioned (1)-(11),-   (39) a screening method of a differentiation inducer for tissue or    cell, which comprises the following steps (i)-(iii):-   (i) a step for bringing a test substance into contact with the rat    embryonic stem cell of any of the aforementioned (1)-(11),-   (ii) a step for evaluating the presence or absence or the extent of    the differentiation of the rat embryonic stem cell,-   (iii) a step for judging whether or not the test substance is a    substance associated with differentiation induction, based on the    evaluation results of the above-mentioned (ii),-   (40) a screening method of a substance acting on the differentiation    induction of tissue or cell, which comprises the following steps    (I)-(III):-   (I) a step for bringing a test substance into contact with the rat    embryonic stem cell of any of the aforementioned (1)-(11),-   (II) a step for culturing the rat embryonic stem cell of the    aforementioned (I) under the conditions allowing differentiation    induction of the embryonic stem cell, and evaluating the presence or    absence or the extent of the differentiation thereof,-   (III) a step for judging whether or not the test substance is a    substance acting on the differentiation induction of tissue or cell,    based on the evaluation results of the above-mentioned (II),-   (41) a use of the rat embryonic stem cell of any of the    aforementioned (1)-(11) in the production of a genetically modified    rat,-   (42) the use of the aforementioned (41), wherein the genetically    modified rat is either of a chimeric rat, knockout rat, knockin rat,    transgenic rat and knockdown rat,-   (43) a production method of a genetically modified rat, which    comprises performing a process comprising the following steps    (X)-(Z):-   (X) a step for introducing a desired gene into the rat embryonic    stem cell of any of the aforementioned (1)-(11),-   (Y) a step for preparing an egg for transplantation comprising the    rat embryonic stem cell into which the gene was introduced,-   (Z) a step for transferring the oocytes for transplantation into a    pseudopregnant female rat to produce an offspring rat,-   (44) a genetically modified rat produced by the production method of    the aforementioned (43),-   (45) the rat of the aforementioned (44), which is either of a    chimeric rat, knockout rat, knockin rat, transgenic rat and    knockdown rat,-   (46) a rat embryonic stem cell obtained by performing a process    comprising the following steps (A)-(D), under the culture conditions    using a culture medium substantially free of serum:-   (A) a step for dissociating an inner cell mass formed by the culture    of rat blastocysts, remaining a state of cell aggregate,-   (B) a step for culturing primary embryonic stem cells resulting from    the culture of the dissociated inner cell mass until it can be    passaged,-   (C) a step for dissociating the primary embryonic stem cells, which    have become capable of being passaged, remaining a state of cell    aggregate, followed by passaging and culturing the same,-   (D) a step for further passaging and culturing the cells to    establish a embryonic stem cell, and characterized by having the    following properties (a)-(j):-   (a) expressing Oct3/4 gene and Nanog gene,-   (b) positive for alkaline phosphatase activity,-   (c) having an embryoid body forming ability,-   (d) expressing SSEA (Stage-Specific Embryonic Antigen)-1 and SSEA-4,-   (e) having the same number of chromosomes as does a normal rat cell,-   (f) capable of being subcultured and holding the undifferentiated    state,-   (g) having in vitro pluripotency,-   (h) having a potential to differentiate to cells of three embryonic    germ lineages,-   (i) having teratoma formation ability,-   (j) having an ability to produce a chimeric rat,-   (47) the rat embryonic stem cell of the aforementioned (46), further    having the property:-   (k) differentiating by culture in the presence of 20% serum, and-   (48) the embryonic stem cell of the aforementioned (46) or (47),    which is derived from either strain of Wistar Kyoto strain (WKY),    Wistar Hannover GALAS strain (WHG) and Brown Norway strain (BN).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of the rat blastocysts (late stage) used for theestablishment of rat ES cells.

FIG. 2 is a photograph of the normal mouse embryonic fibroblasts (feedercells) treated with mitomycin C used for the establishment of rat EScells.

FIG. 3 is a drawing showing the results of the investigation on thenecessity of the addition of rat LIF after the establishment of rat EScells. At beginning the cells are at passage 5. Upon 3 passages usingculture media supplemented with each concentration of rLIF from the sameculture dish, the positive ratios of alkaline phosphatase activity wereexamined, which results are shown.

FIG. 4 shows photographs that show the results of study on the presenceand absence of serum in a culture medium. (A) is a photograph showingthe formation of a rat inner cell mass when 20% serum replacementreagent (KSR) is used, (B) is a photograph showing the formation of arat inner cell mass when 20% fetal bovine serum is used, (C) is aphotograph of the morphology of the culture when the rat inner cell massproduced using 20% serum replacement reagent (KSR) is cultured using aculture medium for rat ES cells supplemented with 20% serum replacementreagent (KSR) and feeder cells, and (D) is a photograph of themorphology of the culture when the rat inner cell mass produced using20% fetal bovine serum is cultured using a culture medium for rat EScells supplemented with 20% fetal bovine serum and feeder cells,respectively.

FIG. 5 shows photographs that show the results of comparison of the caserat ES cells are passaged in a state of aggregate consisting of 5-20cells (a) and the case they are passaged after being completely made tothe single cells with trypsin-EDTA solution. A spontaneousdifferentiation occurs by making them to the single cells.

FIG. 6 is a drawing showing the outlines of a method of rat ES cellestablishment.

FIG. 7 is a photograph that shows the formation of inner cell masses byculturing zona pellucida-removed rat blastocysts using feeder cells anda culture medium for rat ES cell establishment. The arrows show theinner cell masses formed.

FIG. 8 is a photograph of primary rat ES cells that have appeared byculturing rat inner cell masses using a culture medium for rat ES cellsand feeder cells.

FIG. 9 is a photograph of rat ES cells 7 days after the appearance ofthe ES cell colonies, which has become the state capable of passage.

FIG. 10 is a photograph of cell population of rat ES cells (establishedrat ES cells), which have become capable of stable proliferation andpassage by use of a culture medium for rat ES cells and feeder cells.

FIG. 11 shows photographs showing the morphology of established rat EScells. a) is at 100-fold, b) is at 200-fold and c) is at 400-foldmagnification.

FIG. 12 shows photographs of RT-PCR that demonstrates the expression ofmarker genes for undifferentiated state in established rat ES cells. Thepresence or absence of the expression of Oct 3/4 gene and Nanog gene,which are typical marker genes for undifferentiated state in ES cells,were assayed. The expression of β-actin gene was also analyzed as acontrol.

FIG. 13 is a photograph showing an alkaline phosphatase activity ofestablished rat ES cells, which activity is one of the typical indexesdemonstrating that they have an ability to keep undifferentiated state.

FIG. 14 is a photograph showing the results of the analysis for embryoidbody-forming ability of established rat ES cells. About 20 days later,many of embryoid bodies beating like cardiac muscle (arrow) wereconfirmed.

FIG. 15 show photographs presenting the results of immunostaining using4 kinds of marker antibodies ((A): SSEA-1, (B): SSEA-4, (C): TRA-1-60,(D): TRA-1-81) which give an indication that the established cells areES cells.

FIG. 16 is a photograph showing the analysis of the chromosome number ofthe established rat ES cell using G-banding method, which indicates thesame chromosome number (2n=42) as that of normal rat cell.

FIG. 17 is a graph showing the relationship between the passage numberof established rat ES cells and their ability to keep undifferentiatedstate (alkaline phosphatase activity). The staining for alkalinephosphatase activity was started at passage 5, and then performed afterevery 5 passages. The vertical axis shows the number of positive ES cellcolonies divided by the number of total ES cell colonies in percentage.The horizontal axis shows the passage number.

FIG. 18 shows photographs demonstrating that established rat ES cellshave a pluripotency in in vitro system. Seven days after initiation ofembryoid body formation, the embryoid bodies were transferred to agelatin-coated culture dish to induce a spontaneous differentiation. (a)a photograph of rat ES cell-derived neuron-like cells, (b) a close-uppicture of the area surrounded by square in Fig. (a), (c) a photographof rat ES cell-derived adipose-like cells, (d) a photograph of rat EScell-derived epidermoid cells.

FIG. 19 shows photographs showing the results of study on the effects ofserum/LIF addition on established rat ES cells. In the figure, “ratLIF+20% FBS” shows the result of the culture under the conditionscontaining rat LIF (rLIF) and 20% serum, “serum-free” shows the resultof the culture under the conditions free of LIF and serum (containing20% KSR), “mouse LIF+serum-free” shows the result of the culture underthe conditions containing mouse LIF (mLIF) and free of serum (containing20% KSR), and “rat LIF+serum-free” shows the result of the culture underthe conditions containing rat LIF (rLIF) and serum free (containing 20%KSR), respectively.

FIG. 20 shows photographs showing the results that ES cells wereestablished form each rat (WKY, WHG and BN). Upper figure: photographsof inner cell masses formed from each rat blastocysts. In the figure,the arrows show inner cell masses formed. Lower figure: photographs ofestablished ES cells at passage 5. In the figure, WKYrES shows ES cellsestablished from WKY rat, WHGrES shows ES cells established from WHG ratand BNrES shows ES cells established from BN rat, respectively.

FIG. 21 shows photographs of RT-PCR demonstrating that the establishedrat ES cells (WKY rat ES cells) express ES cell-marker genes. The geneexpression of each factor of typical marker genes for ES cells, Oct3/4,Rex-1, Nanog and ERas was analyzed. The expression of β-actin gene wasalso analyzed as a control. Lane 1: undifferentiated rat ES cells, Lane2: mouse fibroblasts, Lane 3: no template DNA (control). RNA wasextracted from each samples and RT-PCR was performed.

FIG. 22 shows photographs of RT-PCR demonstrating that embryoid bodiesformed from established rat ES cells (WHG rat ES cells) differentiate tocells of three embryonic germ lineages. In the figure, the name of eachmaker gene is shown in the left side: TUJ1 shows beta3-tublin, MSI-1shows musashi-1, GFAP shows glial fibrillary acidic proteins alpha, CCCshows cardiac dihydropyridine-sensitive calcium channel protein, ANFshows atrial natriuretic factor, ALB shows albumin, TDO shows tryptophan2,3-dioxygenase, TAT shows tyrosine aminotransferase, G6P showsglucose-6-phospatase and ACT shows β-actin, respectively. Lane 1:undifferentiated rat ES cells, Lane 2: rat ES cell-derived embryoidbodies, Lane 3: control (no template DNA), Lane 4: each organ derivedfrom a WHG individual (brain, heart, liver). RNA was extracted from eachsamples and RT-PCR was performed.

FIG. 23 shows photographs demonstrating that teratomas were formed bysubcutaneously implanting the established rat ES cells (WHG rat EScells) to mice, and that the teratomas have three embryonic germ lineagestructures. a): a photograph of mice in which teratomas were formed bysubcutaneously implanting WHG rat ES cells to immunodeficient mice. Thelumps at the necks are teratomas. b): a photograph of the dissectedteratomas. In the figure, the size bar shows 1 cm. c): histologies of ateratoma sectioned and stained with hematoxylin/eosin. The upper leftshows gland structure, the upper right shows vascular endothelium-likestructure, the lower left shows intestinal tract-like structure and thelower right shows osteocyte-like structure, respectively.

FIG. 24 shows photographs of genomic PCR demonstrating that EGFP isexpressed in almost tissues in a chimeric rat produced using establishedgene-introduced rat ES cells (pCAG-EGFP/WHGrES cells). The upper figureshows the results in the chimeric rat produced using pCAG-EGFP/WHGrEScells, the lower figure shows the results in a wild-type rat. Lane 1:brain, Lane 2: thymus, Lane 3: heart, Lane 4: esophagus, Lane 5: lung,Lane 6: stomach, Lane 7:pancreas, Lane 8: small intestine, Lane 9: largeintestine, Lane 10: liver, Lane 11: spleen, Lane 12: kidney, Lane 13:testis, Lane 14: blood vessel, Lane 15: muscle. Chromosome DNA wasextracted from each tissue, and genomic PCR was performed.

FIG. 25 shows photographs of each tissue (kidney, skeletal muscle andstomach) of a GFP chimeric rat sectioned and immunostained with anti-GFPantibody.

BEST MODE FOR EMBODYING THE INVENTION

The present invention provides rat ES cells that could not be obtainedby conventional methods for the first time.

The rat ES cells of present invention can be established and produced byperforming a process comprising the following steps (A)-(D), under theconditions using a substantially serum free culture medium:

-   (A) a step for dissociating an inner cell mass formed by the culture    of rat blastocysts, remaining a state of cell aggregate,-   (B) a step for culturing primary embryonic stem cells resulting from    the culture of the dissociated inner cell mass until it can be    passaged,-   (C) a step for dissociating the primary embryonic stem cells, which    have become capable of being passaged, remaining a state of cell    aggregate, followed by passaging and culturing the same, and-   (D) a step for further passaging and culturing the cells to    establish a embryonic stem cell.

The characteristics of the method for rat ES cell establishment of thepresent invention is that (1) a substantially serum free culture mediumis used in all of the culture of blastocysts, inner cell masses and EScells, and that (2) in the step for dissociating (exfoliating) innercell masses and ES cells, the cells are not made to single cells butdissociated (exfoliated) with a certain amount of cell aggregatemaintained.

Hereinafter, the method for rat ES cell establishments, the method forcharacterizing established rat ES cells and the method for subculturingrat ES cells and the like of the present invention are explained.

1. Rat

A rat from which the rat ES cells of the present invention are derivedmay be any strain of rat, as long as it can establish ES cells based onthe aforementioned characteristics of the establishment method of thepresent invention. For example, it is selected from the rat strains suchas Wistar Kyoto strain (WKY), Brown Norway strain (BN), Goto-Kakizakistrain (GK), SD strain, F344/Du strain (Fischer), Wistar strain, WistarHannover strain, ACI strain and the like.

2. Feeder Cells

It is preferable to use feeder cells for the establishment and thesubsequent culture of rat ES cells of the present invention. Feedercells may be ones derived from any species available to one of ordinaryskill in the art, and are preferably normal fibroblasts rather thanestablished lines of feeder cells. Specifically, normal mouse embryonicfibroblasts can be mentioned. More specifically, primary cultured cellsof mouse embryonic fibroblasts (normal fibroblasts) between the 12th and16th days of pregnancy can be mentioned. As the normal fibroblasts, forexample, normal fibroblasts of ICR fetal mouse at the 12.5th day areexemplified. The feeder cells can be prepared by a conventional method.Commercially available products (mouse fibroblasts; Asahi Techno GlassCorporation, etc.) can be also utilized. It is preferable to use thefeeder cells inactivated by the treatment with mitomycin C and the like.

3. Culture Medium

For the production (establishment and culture) of the rat ES cells ofthe present invention, a substantially serum free culture medium isused. The “substantially serum free” used herein means not to containserum in an amount that rat ES cell loses the properties as an ES cell(e.g., becomes negative for alkaline phosphatase activity) due to theeffect of serum. Specifically, it means that serum concentration is 10%or less, preferably 5% or less, more preferably 2% or less. Morepreferably, a serum-free medium is used. In this case, it is necessaryto add a reagent to replace serum. Specifically, a serum replacementreagent (KSR: GibcoBRL) and the like are used. The serum replacementreagent is preferably used in a concentration of about 20%.

As regards rat leukemia inhibitory factor (rLIF), it is preferable touse a culture medium without rat LIF (rLIF) in the steps for theformation and separation of inner cell masses from blastocytes. On theother hand, in the steps after the formation of inner cell masses(including the culture and passage of established rat ES cells), anrLIF-containing culture medium is preferably used. As regards theconcentration, it is preferable to add 100 units or more of rLIF per 1ml of culture medium, and more preferable to add about 1000 units ormore of rLIF. A commercially available product (Chemicon, Inc.) can beutilized as the rLIF.

As other components in the culture medium, components conventionallyused for the culture of ES cells are appropriately contained incombination in the scope of one of ordinary skill in the art.

A specific composition of the culture medium is exemplified in thefollowing.

1) Culture Medium for Rat ES Cell Establishment

A culture medium used in the steps from blastocysts to inner cell massformation is referred to as “culture medium for rat ES cellestablishment”.

(Specific Example of Composition)

Dulbecco's modified Eagle medium/F12 (Ashahi Techno Glass, Tokyo, Japan)380 ml

0.2M L-glutamine 5 ml

serum replacement reagent (KSR: Gibco BRL, Funakoshi, Tokyo, Japan) 100ml

non-essential amino acids (Gibco BRL, Funakoshi, Tokyo, Japan) 5 ml

Antibiotic-Antimicrotics solution (Gibco BRL, Funakoshi, Tokyo, Japan) 5ml

100 mM Na-pyruvate 5 ml

0.1M β mercaptoethanol 0.5 ml

2) Culture Medium for Rat ES Cells

A culture medium used in the culture after inner cell mass formation(including culture of established rat ES cells) is referred to as“culture medium for rat ES cells”.

(Specific Example of Composition)

Dulbecco's modified Eagle medium/F12 (Ashahi Techno Glass, Tokyo, Japan)375 ml

0.2M L-glutamine 5 ml

serum replacement reagent (KSR: Gibco BRL, Funakoshi, Tokyo, Japan) 100ml

non-essential amino acids (Gibco BRL, Funakoshi, Tokyo, Japan) 5 ml

100× nucleoside stock solution 5 ml

(adenosine 4 mg, guanosine 4.25 mg, cytidine 3.65 mg, uridine 3.65 mg,thymidine 1.2 mg)

Antibiotic-Antimicrotics solution (Gibco BRL, Funakoshi, Tokyo, Japan) 5ml

100 mM Na-pyruvate 5 ml

0.1M β mercaptoethanol 0.5 ml

1000 U rat leukemia inhibitory factor (rLIF)

Of these, rat leukemia inhibitory factor (rLIF) is preferably added andmixed just before use.

4. Culture Conditions

The temperature of cell culture in the production (establishment andculture) of rat ES cells of the present invention may be within therange of 35° C.-37.5° C., is preferably 37° C. The culture is carriedout in a 5% CO₂ incubator used for a typical culture.

5. Method for Rat ES Cell Establishment

A specific example of the method for rat ES cell establishment of thepresent invention is shown in the following.

1) Oocyte (Embryo in Blastocyst Stage) Sampling

A rat for oocyte sampling is selected from the rat strains such as theaforementioned Wistar Kyoto (WKY) strain, Brown Norway (BN) strain,Goto-Kakizaki (GK) strain, SD strain, F344/Du (Fischer) strain, Wistarstrain, Wistar Hannover strain and ACI strain. A rat within the range of8 to 40 weeks old can be used, preferably a 10 to 20-week-old rat isused, more preferably a 10 to 12-week-old rat is used.

Oocyte sampling may be carried out by a conventional method known to oneof ordinary skill in the art. Specifically, rats are naturally crossed,the female rat for oocyte sampling is sacrificed to excise an uterusabout 3 days after vaginal plug detection. This uterus is perfused witha suitable medium to recover fertilized oocytes (embryos). The culturemedium used herein includes, for example, mw medium (640.0 mg/100 mlNaCl, 35.6 mg/100 ml KCl, 16.2 mg/100 ml KH₂PO₄, 29.4 mg/100 mlMgSO₄-7H₂O, 190.0 mg/100 ml NaHCO₃, 100.0 mg/100 ml glucose, 2.5 mg/100ml Na-pyruvate, 46.0 mg/100 ml Ca-lactate, 5.0 mg/100 ml streptomycin,7.5 mg/100 ml penicillin, 0.5% phenrol red (0.2 ml), 20 mM beta-ME (10μl), 100 mM EDTA-2Na (10 μl), 300.0 mg/100 ml BSA), M2 medium (0.251 g/Lcalcium chloride-2H₂O, 0.143 g/L magnesium sulfate, 0.356 g/L potassiumchloride, 0.162 g/L potassium phosphate, 5.532 g/L sodium chloride, 4.0g/L albumin, 1.0 g/L D-glucose, 4.969 g/L HEPES, 0.01 g/L phenol red-Na,0.036 g/L pyruvic acid-Na, 0.35 g/L sodium bicarbonate, 0.06 g/Lpenicillin G, 0.05 g/L streptomycin sulfate, 4.349 g/L D,L-lactic acid)and the like.

The recovered embryos are cultured in a culture medium such as mwmedium, M2, M16, and the like.

By this culture, the development proceeds from fertilized oocytes(embryos) through moluras to blastocysts (embryos in blastocyst stage).To promote the development to this stage, the culture is typicallyperformed in a 5% CO₂ incubator at 37° C. overnight. It can be confirmedby microscopic observations that the development has proceeded toblastocyst stage. Preferably, the development proceeds up to the lateblastocyst stage.

2) Formation and Separation of Inner Cell Masses

The blastocysts obtained in the aforementioned 1) are confirmedmicroscopically, and zona pellucidas are removed. The zona pellucidas isremoved using Acidic Tyrode (pH 2.5), hyaluronidase, pronase and thelike. Then, feeder cells treated with mitomycin C are sown ontogelatin-coated culture dishes, 5-10 zona pellucida-removed ratblastocysts are transferred to each of the dishes, and the culture isstarted using a culture medium for rat ES cell establishment.

Between the 1st and 4th days of the culture, zona pellucida-removed ratblastocysts (late stage) adhere to the feeder cells. On 5-10 days afteradhesion, an inner cell mass appeared from the blastocysts ismechanically separated using a 200 μl of pipette and the like. Thisseparated inner cell mass is transferred to a sterilized tube and thelike containing a culture medium for rat ES cell establishment, anddissociated until it becomes cell aggregates consisting of about 5-20cells. At the time, the inner cell mass is not dissociated with aprotease such as trypsin-EDTA and the like but is mechanicallydissociated using a pipette and the like. In addition, it should beavoided to carry out the dissociation until single cells, and thedissociation is performed to the extent that cell aggregates consistingof about 5-20 cells remain as mentioned above. Being in a state thatcell aggregates remain can be confirmed microscopically.

3) Establishment of ES Cells

In the gelatin-coated culture dishes wherein the feeder cells are sown,inner cell masses dissociated in the aforementioned 2) are cultured in aculture medium for rat ES cells. Primary ES cell colony usually appearsbetween the 2nd and 4th days of the culture. The appearance of theprimary ES cell colony can be confirmed by microscopic observations (theappeared ES cells are referred to as “primary ES cells”). By continuingthe culture about 5-10 days thereafter, the primary ES cell colonybecomes in a state capable of being passaged. The “state capable ofbeing passaged” used herein means a state wherein the number of cellsconstituting the primary ES cell colony formed has reached approximately200-600, and each cellular interval has become tight. Whilemicroscopically confirming that it has such morphology, the ES cellcolony is separated using a 200 μl of pipette and the like. Thisseparated ES cell colony is transferred to a sterilized tube and thelike containing a culture medium for rat ES cells, and dissociated untilit becomes cell aggregates consisting of about 5-20 cells. In thisstage, it should also be avoided to carry out the dissociation untilsingle cells, and the dissociation is performed to the extent that cellaggregates remain as mentioned above. Also in this stage, it isdesirable that the cells are not dissociated with a protease such astrypsin-EDTA and the like but is mechanically dissociated. Thedissociated ES cell colony is subjected to a primary culture (cells atpassage 1) in a culture medium for rat ES cells in a gelatin-coatedculture dish wherein the feeder cells are sown. An ES cell colonyappears about 2-4 days and becomes in a state capable of being passagedabout 5-10 days after the beginning of the culture.

The passage of the cells thereafter can be performed within the commonknowledge of one of ordinary skill in the art, as long as maintaining astate that the cells are not made to single cells, namely a state thatcell aggregates consisting of about 5-20 cells remain. It is desirablethat cell dissociation is performed in a way minimizing a treatment witha protease such as trypsin-EDTA and the like and using a physical means.A specific example is shown in the following.

After removing the culture medium, the ES cell colony that has becomecapable of passaged in the above are washed with PBS(−) at roomtemperature and the entire surface thereof is coated with 2.5% trypsinpreviously incubated at 37° C. The amount of trypsin is preferably about500 μl or more per 60 mm dish. The solution is removed immediately afterthe full coating with trypsin. When microscopically confirming a statethat 70% or more of whole the ES cell colony is coming unstuck from thefeeder cells, the trypsin treatment is immediately stopped. To stop thetrypsin treatment herein, for example, it is easy to add a culturemedium containing 10% fetal bovine serum. Alternatively, the treatmentcan be stopped by adding a large amount of serum-free culture medium andthe like to dilute the trypsin concentration. Then, the ES cell colonyis further unstuck mechanically using a 5 ml of pipette and the like,the cell suspension is centrifuged (for about 3 minutes at roomtemperature, 1000 rpm) to separate the cells and the culture medium andonly the cells are recovered. The cells are suspended in a culturemedium for rat ES cells, after microscopically confirming a state thatthe cells form aggregates of 5-20 cells rather than becoming fullysingle cells, the cells are transferred to a gelatin-coated culturedishes wherein the feeder cells are sown and cultured (cells at passage2).

After that, since the cells become in a state capable of passaged everyabout 5-10 days, they can be passaged and cultured by the samesubculture method as in the passage of the cells at passage 1 (passageto the cells at passage 2).

It is deemed that the ES cell is established when the numbers of thecell aggregates and the cells constituting the cell aggregates becomeconstant. Specifically, the cell at passage 3 or later, desirablypassage 5 or later can be deemed as the established rat ES cell. Whenestablished rat ES cells are supplied as a commercial product, cells atpassage 3 or later, preferably passage 5 or later, more preferablypassage 10 or later are the objects for commercialization, in order tomaintain a stable supply from a single line.

6. Rat ES Cell of the Present Invention

The rat ES cell of the present invention is characterized by having thefollowing properties (a)-(j):

-   (a) expressing Oct3/4 gene and Nanog gene,-   (b) positive for alkaline phosphatase activity,-   (c) having an embryoid body forming ability,-   (d) expressing SSEA (Stage-Specific Embryonic Antigen)-1 and SSEA-4,-   (e) having the same number of chromosomes as does a normal rat cell,-   (f) capable of being subcultured and holding the undifferentiated    state,-   (g) having in vitro pluripotency,-   (h) having a potential to differentiate to cells of three embryonic    germ lineages,-   (i) having teratoma formation ability, and-   (j) having an ability to produce a chimeric rat.

The present invention provides a rat ES cell retaining all theproperties as an ES cell shown in the aforementioned (a)-(j) for thefirst time. It can be analyzed by the following methods that theestablished rat ES cell retains the properties as an ES cell, that isthe properties as an ES cell maintaining an undifferentiated state(totipotency).

1) Expression of Markers for Undifferentiated State

Oct3/4 (Okamoto, K. et al., Cell, 60: 461-472 (1990), Scholer, H. R. etal., EMBO J. 9: 2185-2195 (1990)) and Nanog (Mitsui, K. et al., Cell,113: 631-642 (2003), Chambers, I. et al., Cell, 113: 643-655 (2003)) areknown as critical factors defining a cell as an ES cell. The expressionof these genes can be determined by RT-PCR using rat Oct3/4- andNanog-specific primers. As the rat Oct3/4- and Nanog-specific primersused herein, the primers described in the Examples (Oct3/4:5′-ATGGACTACCCAGAACCCCAG-3′ (SEQ ID NO:3), 5′-TTACAGGAGCTGCAGTTATAC-3′(SEQ ID NO:4), Nanog: 5′-TAGCCCTGATTCTTCTAGCA-3′ (SEQ ID NO:5),5′-TTTGCTGCAACGGCACATAA-3′ (SEQ ID NO:6)) are exemplified.

2) Alkaline Phosphatase Activity

An undifferentiated ES cell expresses an alkaline phosphatase in largeamounts. The expression of the alkaline phosphatase can be easilydetermined using various commercially available alkaline phosphatasedetection kits. The detection kits include, for example, ALP tissuestaining kit (Sigma) and Vector Red Alkaline Phosphatase Substrate Kit I(Funakoshi) and the like.

3) Embryoid Body Forming Ability

It is known that an embryoid body is formed by culturing an ES cellusing a non-coated culture dish under conditions without feeder cellsand LIF (Roy, S. et al., Mol. Cell. Biol., 18: 3947-3955 (1998)). Theembryoid body formation can be confirmed by microscopic observations ofthe appearance of a spherical body formed by cell aggregation after ratES cells are cultured using a culture medium for rat ES cells withoutLIF in a non-coated culture dish for about 7 days to 14 days.

4) Expression of Cell Surface Antigen

As one of the indexes identifying the differentiation of pluripotentstem cells including ES cell, the detection of cell surface antigenswhose expression amounts change specifically in the differentiationstage can be mentioned. The rat ES cell of the present inventionexpresses SSEA (stage-specific embryonic antigen)-1 and SSEA-4. Theexpression of the cell surface markers can be evaluated by animmunostaining using ES Cell Characterization Kit (Funakoshi).

5) Chromosome Number

It can be confirmed by analyzing the chromosome number by G-bandingmethod (Sumner, A. T., Cancer Genet Cytogenet. 6: 59-87 (1982)) that theestablished rat ES cell is a normal ES cell maintaining the chromosomenumber (2n=42) of rat from which it originates.

6) Maintenance of Undifferentiated State

The established ES cell can be subcultured with maintenance ofundifferentiated state. The rat ES cell established by the presentinvention has a superior characteristic that it can be subcultured untilat least 35 passages. The maintenance of undifferentiated state can beconfirmed by performing a subculture according to the subculture methodof the rat ES cell of the present invention (the aftermentioned 7.), anddetermining the aforementioned alkaline phosphatase activity and thelike.

7) Pluripotency

ES cell spontaneously differentiates into various cells through anembryoid body by culturing it under conditions without feeder cells andLIF. This property of ES cell can be observed by forming an embryoidbody by the method described in the aforementioned 3), then transferringthe embryoid body to a gelatin-coated culture dish and culturing it forabout 7-14 days. The appearance of neuron-like cell, adipose-like cellor epidermoid cell or the like can be confirmed by the characteristicmorphology of the each cell.

8) Differentiation Potential to Cells of Three Embryonic Germ Lineages

ES cell has a potential differentiating to cells of three embryonic germ(endoderm, mesoderm, ectoderm) lineages. This property of ES cell can beconfirmed by extracting RNA from an embryoid body (embryoid body whereina myocardium-like cell appears) formed by the method described in theaforementioned 3), and analyzing the expression of each marker gene forectodermal cell (e.g., neuron), mesodermal cell (e.g., cardiomyocyte)and endodermal cell (e.g., hepatocyte) by RT-PCR.

The neuron markers include Nestin, TUJ1 (beta3-tublin), MSI-1(musashi-1), GFAP (glial fibrillary acidic proteins alpha) and the like.The cardiomyocyte markers include CCC (cardiac dihydropyridine-sensitivecalcium channel protein), ANF (atrial natriuretic factor), KvLQT1 andthe like. The hepatocyte markers include ALB (albumin), TDO (tryptophan2,3-dioxygenase), TAT (tyrosine aminotransferase), G6P(glucose-6-phospatase) and the like.

9) Ability of Teratoma Formation

The transplantation of ES cells to a homologous or innateimmunodeficient heterologous animal can lead to a teratoma formation.The teratoma used herein is the designation of a mixed tumor whereinvarious tissues derived from three embryonic germs, endoderm, mesodermand ectoderm, are randomly present in a tumor. The formation of theteratoma can be confirmed by transplanting rat ES cells to the subdermicand the like of a homologous animal or a heterologous animal with aprimary immune deficiency, and macroscopically observing the presence ofa bunchy body after several months. It can be confirmed that theteratoma formed has three embryonic germ structures by sectioning theexcised teratoma, staining with hematoxylin/eosin and observing themorphology of the tissues and cells microscopically.

10) Chimeric Rat-Producing Ability

A chimeric rat can be produced by introducing ES cells into a homologousor heterologous rat. The production of the chimeric rat can be carriedout by, for example, the following method.

To facilitate confirmation of the production of the chimeric rat, amarker gene (e.g., GFP, β-gal, luciferase, etc.) may be previouslyintroduced into the rat ES cell of the present invention. Specifically,a recombinant rat ES cell incorporating a vector containing such markergene in the ES cell chromosome is established by incorporating theaforementioned vector into the rat ES cell chromosome by anelectroporation method and the like, followed by selection in a culturemedium supplemented with a drug. The recombinant rat ES cells are, forexample, transplanted into a blastocoele of a rat blastocyst or intomorula stage or 16-cell stage embryo by a microscopic manipulation anddeveloped with an inner cell mass or as a part of an inner cell mass(microinjection method: Gordon J. W. et al., Proc. Natl. Acad. Sci.USA., 77: 7380-7384 (1980)). Alternatively, zona pellucidas are removedfrom two 8-cell embryos and the embryos are co-cultured with theaforementioned recombinant rat ES cells to form an aggregate. When theresulting aggregate is cultivated, one blastocyst is obtained (cellaggregate method: Dvorak P. et al., Int. J. Dev. Biol., 39: 645-652(1995)). The embryo (egg for transplantation) obtained above istransplanted in the uterus of a pseudopregnant female rat prepared bynatural crossbreeding with a male rat after vasoligation treatment anddeveloped, whereby a chimeric rat can be produced.

That the obtained chimeric rat has cells and tissues derived from an EScell, or an established rat ES cell has a chimeric rat-producing abilitycan be confirmed by a general method known to those of ordinary skill inthe art. For example, it can be confirmed by genomic PCR using genomicDNA extracted from various tissues of chimeric rat as a template andmarker gene (marker gene introduced into ES cell)-specific primers.Moreover, differentiation of ES cell to the cell of each tissue lineagecan be confirmed, for example, by sectioning each tissue of a chimericrat, and detecting the presence of a marker gene expression product(marker protein) based on the properties of the marker protein used.

11) Differentiation by Culture in the Presence of Serum

Moreover, the rat ES cell of the present invention has a property thatit differentiates by culture in the presence of 20% serum. Preferably,the rat ES cell of the present invention differentiates by culture inthe presence of 10% serum, more preferably by culture in the presence of5% serum. The differentiation of rat ES cell can be confirmed by thedisappearance of alkaline phosphatase activity, or disappearance ofexpression of ES cell marker genes such as Oct3/4, Nanog and the like.

7. Subculture Method of Rat ES Cell

The rat ES cell of the present invention can be subcultured whilemaintaining an undifferentiated state. The rat ES cell of the presentinvention can be passaged within the range of technical common knowledgeof those of ordinary skill in the art, as long as the cells aremaintained in a non-single-cell state, namely, a state of cell aggregateconsisting of about 5-20 cells. It is desirable that a treatment byproteinase such as trypsin-EDTA and the like is applied as little aspossible, and cell dissociation is carried out by a physical means.Specific examples are shown in the following.

After removing the culture medium, the established ES rat cells thathave become capable of being passaged are washed with PBS(−) at roomtemperature and the entire surface thereof is coated with 2.5% trypsinpreviously incubated at 37° C. The amount of trypsin is preferably about500 μl or more per 60 mm dish. The solution is removed immediately afterthe full coating with trypsin. When microscopically confirming a statethat 70% or more of whole the ES cell colony is coming unstuck from thefeeder cells, the trypsin treatment is immediately stopped. To stop thetrypsin treatment herein, for example, it is easy to add a culturemedium containing 10% fetal bovine serum. Alternatively, the treatmentcan be stopped by adding a large amount of serum-free culture medium andthe like to dilute the trypsin concentration. Then, the ES cell colonyis further unstuck mechanically using a 5 ml of pipette and the like,the cell suspension is centrifuged (for about 3 minutes at roomtemperature, 1000 rpm) to separate the cells and the culture medium andonly the cells are recovered. The cells are suspended in a culturemedium, after microscopically confirming a state that the cells formaggregates of 5-20 cells rather than becoming fully single cells, thecells are transferred to a gelatin-coated culture dishes wherein thefeeder cells are sown and cultured. Thereafter, the passageable stateoccurs every about 5-10 days, and subculture is conducted in the samemanner.

For the culture of passaged cells, a culture medium substantially freeof serum is used. The “substantially free of serum” used herein meansnot to contain serum in an amount that rat ES cell loses the propertiesas an ES cell (e.g., becomes negative for alkaline phosphatase activity)due to the effect of serum. Specifically, it means that serumconcentration is 10% or less, preferably 5% or less, more preferably 2%or less. More preferably, a serum-free medium is used. In this case, itis necessary to add a reagent to replace serum. Specifically, a culturemedium containing a serum replacement reagent (KSR: GibcoBRL) and thelike are used. The serum replacement reagent is preferably used in aconcentration of about 20%.

The culture medium to be used for the culture of the aforementionedpassaged cells desirably contains rLIF. As the concentration of rLIF,rLIF is preferably added at not less than 100 units, and more preferablyabout 1000 units, or not less than that, per 1 ml of the culture medium.

As mentioned above, a culture medium for subculture of the rat ES cellof the present invention preferably contains a serum replacement reagentand rLIF, and the culture medium for rat ES cells described in theaforementioned 3.-2) is used effectively.

8. Rat ES Cell Culture Kit

The present invention provides a culture kit for cultivating the rat EScell of the present invention. To be specific, the present inventionprovides a rat ES cell culture kit containing a serum replacementreagent (KSR) and rLIF as components. While the serum replacementreagent and rLIF in a mixture may be sealed in a single container, theyare each preferably sealed in a separate container. As KSR, onemanufactured by Gibco BRL can be used, and as rLIF, one manufactured byChemicon can be used.

The aforementioned kit can further contain a rat ES cell of the presentinvention as a component. When the rat ES cell of the present inventionis supplied as a commercial product, the cell at passage 3 or later,preferably passage 5 or later, more preferably passage 10 or later arethe objects for commercialization. While the rat ES cell can becommercialized by itself, it may be commercialized as one component ofthe culture kit of the present invention as mentioned above.

The aforementioned kit can further contain a feeder cell as a component.The feeder cell may be one derived from any species available to one ofordinary skill in the art, and is preferably normal fibroblast ratherthan an established line of feeder cell. Specifically, primary culturedcells of mouse embryonic fibroblasts (normal fibroblasts) between the12th and 16th days of pregnancy can be mentioned. As the normalfibroblasts, for example, normal fibroblasts of ICR fetal mouse at the12.5th day are exemplified. The feeder cells can be prepared by aconventional method. Mouse embryonic fibroblasts (Asahi Techno GlassCorporation), etc. can be also utilized.

The production methods (establishment method, subculture method) of EScell, and culture medium and culture kit of ES cell as mentioned aboveare applicable to rat ES cell as well as other animal species (exceptmouse).

Mouse ES cell colony rises like a dome, with generally indefinite cellboundary between the adjacent cells. It is glossy. In contrast, thecolony of primate ES cells is flattened, with no gloss as observed inmouse ES cell. Moreover, the boundary between cells is clearer than inmouse ES cells. It has been clarified that the rat ES cells establishedin the present invention form a flat colony, free of high glosspossessed by mouse ES cell, and show morphology closer to that ofprimate ES cell than mouse. Therefore, the production methods(establishment method, subculture method) of ES cell of the presentinvention, and culture medium and culture kit of ES cell as mentionedabove are applicable to ES cell of primates.

9. Cell Obtained by Differentiation Induction of Rat ES Cell

The present invention provides a differentiation induction method of ratES cell of the present invention, and a cell obtained by differentiationinduction.

As known in mouse and human ES cells, ES cell can be induced todifferentiate into various cells by culturing ES cell under theconditions free of feeder cell and LIF (Roy. S. et al., Mol. Cell.Biol., 18: 3947-3955 (1998)). In addition, it can be induced todifferentiate into various cells by the action of retinoic acid, cellgrowth factors, glucocorticoids and the like (Kawamorita M. et al., Hum.Cell., 15: 178-182 (2002)). Moreover, differentiation induction can beperformed with an extracellular substrate. Accordingly, similardifferentiation induction can be performed by cultivating the rat EScell of the present invention under the aforementioned conditions. Asthe cells obtained here by differentiation induction of ES cell, forexample, neuron, hematopoietic cell lineage, hepatocyte, vascularendothelial cell, cardiomyocyte and the like can be mentioned.

The cells obtained by differentiation induction of ES cell are useful,for example, as the cells for transplantation in an experimental modelfor a transplantation treatment. In addition, the differentiated cellcan also be used effectively for examining the influence of exposure ofthe cell to nucleic acid, protein or carcinogen, environmental mutagensubstance and the like. To be specific, the influence on the cell can beexamined based on the biological indices such as genetic or epigeneticchanges of the cell, cell transformation, colony formation ability insoft agar medium, changes of canceration indices including infiltrationability, changes of metabolic function, changes of physiologicalfunction, biochemical changes and the like, by the addition of theaforementioned substances.

10. Substance Derived from Rat ES Cell

By establishing the rat ES cell in the present invention, not only ratES cell but also various substances derived therefrom (e.g., cDNAlibrary prepared from mRNA and genomic library of the established rat EScell, or cell extract of rat ES cell and the like) can be provided.Various substances derived from the ES cell of the present invention canbe used effectively for the research of regenerative medicine and studyof molecular level expression analysis, drug discovery research andsafety evaluation test and the like.

As used herein, the cDNA library can be produced using RNA extractedfrom the rat ES cell of the present invention as a template and acommercially available cDNA library construction kit (e.g., CloneMinercDNA Library Construction Kit (Invitrogen), Creator SMART cDNA LibraryConstruction Kit (BD Biosciences) etc.) and the like. In addition, agenomic library can be produced by a conventional method by reference tobasic books such as Molecular Cloning, A Laboratory Manual., T. Maniatiset al. ed., 2nd edition (1989), Cold Spring Harbor Laboratory and thelike. In addition, the cell extract derived from ES cell can be preparedby disrupting the cell by a conventional method, centrifugation in thepresence of a protease inhibitor and the like.

11. Screening Method for Tissue or Cell Differentiation Inducer

The present invention provides a screening method for a tissue or celldifferentiation inducer, which comprises the following (i)-(iii):

-   (i) a step for contacting a test substance with the rat ES cell of    the present invention,-   (ii) a step for evaluating the presence or absence, or the extent of    differentiation of rat ES cell, and-   (iii) a step for judging whether or not the test substance is a    substance associated with differentiation induction, based on the    evaluation results of the above-mentioned (ii).

While the test substance to be subjected to the screening in theaforementioned step (i) is not limited, nucleic acid, peptide, protein,organic compound, inorganic compound and the like can be mentioned. Theaforementioned screening is specifically performed by bringing the testsubstance or a sample containing the substance (test sample) in contactwith the rat ES cell. As such test sample, cell extract, gene (genomic,cDNA) library, RNAi library, antisense nucleic acid, syntheticlow-molecular-weight compound, synthetic peptide, natural compound andthe like can be mentioned. These test samples and test substances arecontacted in a form permitting uptake into the rat ES cell. For example,when the test sample is a nucleic acid, it is introduced into the EScell by microinjection, calcium phosphate, DEAE-dextran or a lipid forgene introduction.

The conditions under which the rat ES cell is brought into contact withthe test substance are not particularly limited as long as the cell isnot killed and the culture conditions (temperature, pH, composition ofculture medium and the like) are suitable for uptake of the testsubstance. Preferably, the aforementioned test substance is added to thecultured rat ES cell under the conditions suitable for differentiationinduction, namely, under the conditions free of feeder cell and LIF.

In the subsequent step (ii), the presence or absence, or the extent ofdifferentiation of rat ES cell is evaluated, and in (iii), whether ornot the test substance is related to the differentiation induction isjudged based on the evaluation results. Differentiation of the rat EScell into a desired tissue or cell can be evaluated, for example, using,as an index, a marker that expresses in a desired tissue or cell. As themarker of the desired tissue or cell, a tissue- or cell-specific antigencan be mentioned. Specifically, as the marker, for example,neuron-specific enolase, glial fibrillary acidic protein, Nestin and thelike can be mentioned as a neural cell marker, S-100 protein, tartaricacid resistant acid phosphatase and the like can be mentioned as acartilage marker. As a muscle marker, desmin, muscle-specific actin andthe like can be mentioned. Such tissue or cell specific marker can bedetected by ELISA, immunostaining and the like, using an antibodyagainst the marker. It is also possible to detect expression of themarker gene by RT-PCR and the like.

12. Screening Method for Substance Acting on Tissue or CellDifferentiation Induction

The present invention provides a screening method of a substance actingon the differentiation induction of tissue or cell, which comprises thefollowing steps (I)-(III):

-   (I) a step for contacting a test substance with the rat ES cell of    the present invention,-   (II) a step for culturing the rat ES cell of the aforementioned (I)    under the conditions allowing differentiation induction of the ES    cell, and evaluating the presence or absence or the extent of the    differentiation thereof, and-   (III) a step for judging whether or not the test substance is a    substance acting on the differentiation induction, based on the    evaluation results of the above-mentioned (II).

As mentioned above, ES cell can be induced to differentiate into variouscells by culturing ES cell under the conditions free of feeder cell andLIF (Roy. S. et al., Mol. Cell. Biol., 18: 3947-3955 (1998)). Inaddition, it can be induced to differentiate into various cells by theaction of retinoic acid, growth factors, glucocorticoids and the like(Kawamorita M. et al., Hum. Cell., 15: 178-182 (2002)). Moreover,differentiation induction can be performed with an extracellularsubstrate. Addition of a test compound (e.g., pharmaceutical productsuch as anti-cancer agent and the like, environmental mutagen and thelike) to the culture system followed by culture enables to evaluate aninfluence of the test substance on normal differentiation. The screeningcan be applied to, for example, the study of the side effects of thepharmaceutical product under development and the like.

As the test substance subjected to the screening in step (I), thosesimilar to the substance of the aforementioned 11. can be mentioned. Forcontact of the test substance with the rat ES cell, the test substancemay be brought into contact with the rat ES cell of the presentinvention and then cultured under the conditions for the differentiationinduction of ES cell, or the rat ES cell may be cultured under theconditions for the differentiation induction and thereafter contactedwith the test substance.

As the culture under the conditions for differentiation induction, underthe aforementioned culture conditions free of feeder cell and LIF,culture conditions where retinoic acid, growth factors, glucocorticoids,extracellular substrate and the like are added to the culture medium andthe like can be mentioned. During the culture process or after theculture under the conditions for differentiation induction, an influenceof the test substance on the differentiation of the rat ES cell isevaluated. The evaluation is desirably conducted based on the comparisonof the extent of the differentiation in the object cell without anaction of the test substance. To be specific, for example,differentiation to functional mature hepatocyte is induced by theaddition of a hepatocyte differentiation inducer (JP 2004-59705 A) to aculture medium, and can be evaluated using a hepatocyte-specificallyexpressing gene (albumin, tryptophan 2,3-dioxygenase etc.) and the likeas an index.

13. Genetically Modified Rat

The rat ES cell of the present invention can be used for the preparationof a genetically modified rat.

Rat is a mammal having an experimentally suitable size of about 10 timesthe size of mouse, and is advantageous in that (1) drug administrationinto the blood vessel in the cell is easy, (2) a surgical ortransplantation test can be performed, (3) a large amount of tissue canbe collected and the like. While many human disease model rats have beenconventionally developed and discovered, since rat ES cell was notestablished, preparation of a genetically modified rat, particularly agenetically modified rat requiring gene targeting such as knockout ratand knockin rat, has been practically impossible. The rat ES cell of thepresent invention has first enabled preparation of such a geneticallymodified rat. By the provision of the rat ES cell of the presentinvention, the genetically modified rat can be produced by the techniquewell known to those of ordinary skill in the art.

Here, the “genetically modified rat” means any genetically modified ratknown to those of ordinary skill in the art, such as chimeric rat,knockout rat, knockin rat, transgenic rat and knockdown rat.

The aforementioned genetically modified rat can be produced by a processincluding the following steps (X)-(Z):

-   (X) a step for introducing a desired gene into the rat ES cell of    the present invention,-   (Y) a step for preparing an oocyte for transplantation comprising    the rat ES cell into which the gene was introduced, and-   (Z) a step for transferring the oocyte for transplantation into a    pseudopregnant female rat to produce an offspring rat.

Knockout rat means a mutant rat wherein a target gene has beenartificially destroyed, and is also called a gene targeting rat. Aknockout rat can be prepared, for example, according to a preparationmethod of a knockout mouse as described in Donehower, A.L. et al.Nature, 356: 215-221 (1992) and the like. Briefly, a vector forhomologous recombination (targeting vector) is constructed based on thegenomic DNA sequence of a target gene. At this time, a drug resistantgene such as a G418 resistant gene, a hygromycin resistant gene and thelike is incorporated as a marker gene for selection of a recombinantclone. The constructed targeting vector is introduced into the rat EScell by an electroporation method and the like. From the obtainedtransfected cells, a colony in which a homologous recombination hasoccurred is selected. The homologously recombined rat ES cell thusobtained is, for example, transplanted into a blastocoele of a ratblastocyst or into morula stage or 16-cell stage embryo by a microscopicmanipulation and developed with an inner cell mass or as a part of aninner cell mass (microinjection method: Gordon J. W. et al., Proc. Natl.Acad. Sci. USA., 77: 7380-7384 (1980)). Alternatively, zona pellucidasare removed from two 8-cell embryos and the embryos are co-cultured withthe aforementioned recombinant rat ES cells to form an aggregate. Whenthe resulting aggregate is cultivated, one blastocyst is obtained (cellaggregate method: Dvorak P. et al., Int. J. Dev. Biol., 39: 645-652(1995)). The embryo (oocyte for transplantation) obtained above istransplanted in the uterus of a pseudopregnant female rat prepared bynatural crossbreeding with a male rat after vasoligation treatment anddeveloped, whereby a chimeric rat can be produced. By crossing thechimeric rat with a wild-type rat, a heterozygous knockout rat can beproduced, and when heterozygous knockout rats are crossed, a homozygousknockout rat can be produced.

The category of the aforementioned knockout rat includes a conditionalknockout rat. The conditional knockout means a system tosite-specifically or time-specifically knockout a gene utilizing aCre/loxP system or a FLP/FRT system. To be specific, a gene to betargeted is substituted by a gene flanked by loxP sequences or FRTsequences, and Cre or FLP protein is supplied to cleave the gene flankedby the aforementioned loxP sequences or FRT sequences (Sternberg N., etal., J. Mol. Biol., 150: 487-507 (1981)).

Knockin rat means a mutant rat in which an artificially preparedexogenous gene having homology to a target gene has been introduced intothe site thereof. The target gene may or may not be destroyed. Forexample, to monitor gene expression, a marker gene such as a lacZ gene,a GFP gene and the like may be introduced or a gene may be exchanged toone in which a mutation has been introduced.

The knockin rat can be produced, for example, according to a preparationmethod of a knockin mouse as described in Pewzner-Jung, Y. et al., J.Immunol., 161: 4634-4645 (1998) and the like. Basically, the rat can beproduced by the same principle as in the aforementioned knockout rat.

Transgenic rat means a rat in which a foreign gene has been artificiallyintroduced. A transgenic animal is conventionally prepared by injectionof a desired gene into the male pronucleus of a fertilized oocyte bymicroscopic manipulation. Therefore, the need for the rat ES cell of thepresent invention is not as high as in the case of the aforementionedknockout rat and knockin rat. However, the rat ES cell of the presentinvention is effectively used for increasing the introduction efficiencyand individual preparation efficiency.

The transgenic rat can be produced, for example, according to thepreparation method of transgenic mouse as described in Yamamoto H. etal., Cancer Res., 62: 1641-1647 (2002) and the like. That is, ES cell issacked with a glass pipette to hold thereon, and an exogenous targetgene solution is directly injected into the nucleus from the other endusing a thin and sharp glass pipette (tip not more than 2 μm). The EScell, into which the solution has been injected, is transferred to aculture system. A strain incorporating the gene is established, afertilized oocyte (morula stage or blastocyst stage embryo) of mouse issacked with a glass pipette to hold thereon, the established ES cell isinjected into the fertilized oocyte with a glass pipette, the oocyte iscultured in a test tube, developed to a certain level, and returned tothe oviduct or uterus of a pseudopregnant female mouse to produce anoffspring mouse.

The category of the aforementioned transgenic rat includes a conditionaltransgenic rat. The conditional transgenic means a system tosite-specifically or time-specifically express a gene utilizing aCre/loxP system or a FLP/FRT system. To be specific, a drug resistantgene is flanked by loxP sequences or FRT sequences, and Cre or FLPprotein is supplied to cleave the gene flanked by the loxP sequences orFRT sequences, whereby the object gene is expressed (Sternberg, N., etal., J. Mol. Biol., 150: 487-507 (1981)).

Knockdown rat means a rat, into which a short double strand RNA (siRNA),which is an intermediate for RNAi, or antisense nucleic acid has beenartificially introduced and expressed, and expression of the target geneis suppressed by the action of the siRNA or antisense nucleic acid.Preparation of such knockdown animal has been enabled based on theestablishment of an expression system of siRNA by a vector system(Science 296: 550-553 (2002), Nature Biotech. 20: 500-505 (2002) etc.).

The knockdown rat can be produced, for example, according to the methoddescribed in Tiscornia, G. et al., Proc. Natl. Acad. Sci. USA. 100:1844-1848 (2003) and the like. Basically, the rat can be produced by thesame principle as in the aforementioned transgenic rat.

The present invention also provides a genetically modified rat producedby the above operation. Here, “genetically modified rat” means anygenetically modified rat known to those of ordinary skill in the art, asmentioned above, such as chimeric rat, knockout rat, knockin rat,transgenic rat and knockdown rat.

EXAMPLES

The present invention is explained in detail in the following byreferring to Examples, which are not to be construed as limitative.

Example 1 Establishment of Rat ES Cell 1) Oocyte Sampling

A female WKY/N (Wistar Kyoto strain, Charles River Laboratories Japan,10-week-old or older) was naturally crossbred, at 3 days after vaginalplug confirmation, the female rat for oocyte sampling was sacrificed andthe uterus was excised. After perfusing with mw medium (640.0 mg/100 ml,NaCl, 35.6 mg/100 ml KCl, 16.2 mg/100 ml KH₂PO₄, 29.4 mg/100 mlMgSO₄-7H₂O, 190.0 mg/100 ml NaHCO₃, 100.0 mg/100 ml Glucose, 2.5 mg/100ml Na-pyruvate, 46.0 mg/100 ml Ca-lactate, 5.0 mg/100 ml Streptomycin,7.5 mg/100 ml Penicillin, 0.5% phenrol red (0.2 ml), 20 mM beta-ME (10μl), 100 mM EDTA-2Na (10 μl), 300.0 mg/100 ml BSA), the embryo wasrecovered, and developed to become blastocyst (late stage) in 5% CO₂incubator (FIG. 1). In the establishment of rat ES cell, since oocytesin the late stage of blastocyst showed the highest establishment (innercell mass formation) efficiency, the oocyte in the late stage ofblastocyst was used as shown above.

2) Preparation of Feeder Cell

As a feeder cell to be used for the establishment and culture of rat EScell, normal fibroblast of fetal mouse ICR at 12.5 days treated withmitomycin C was used (FIG. 2). Before use, a cryopreserved feeder cellwas thawed one day before use, and cultured using STO medium (DMEM 450ml, FBS 50 ml, Antibiotic-Antimicrotics solution 5 ml) and agelatin-coated culture dish (Iwaki, Tokyo, Japan).

3) Consideration of Culture Medium for Rat ES Cell Establishment andCulture Medium for Rat ES Cells (1) Consideration of LIF Addition

An inner cell mass is formed from the rat blastocyst prepared in theaforementioned 1), an ES cell colony is formed from the inner cell mass,and a stabilized rat ES cell is finally established. The culture mediumto be used for a series of these steps was considered.

The necessity of addition of a rat leukemia inhibitory factor (rLIF) toa culture medium during the step of formation of an inner cell mass fromrat blastocyst was first considered. Various concentrations (0-5000units) of rLIF (Chemicon) were added during the step of formation of aninner cell mass from rat blastocyst, and the blastocyst was cultured.The number of the inner cell masses formed thereafter and the number ofthe primary ES cells were observed. As a result, inner cell mass wasformed efficiently without rLIF, which was shown to have varieddepending on the concentration of rLIF added. From such results,LIF-free culture medium was used until the end of the stage of innercell mass formation.

Then, whether or not to add rLIF during the stage of from after theinner cell mass formation to establishment of rat ES cell wasconsidered. As a result of culture in a culture medium containing rLIF(1000 units) after the inner cell mass formation and separation, ES cellcould be established from the inner cell mass at a rate of not less thanabout 50%. Furthermore, the necessity of addition of rLIF after rat EScell establishment was also examined. The rat ES cell was cultured topassage 5 in a culture medium containing 1000 units of rLIF, variousconcentrations of rLIF was added to the rat ES cells (passage 5), andthe cells were subcultured for 3 passages. An ability of the cell tomaintain the undifferentiated state was determined by alkalinephosphatase positive ratio (%) measurement (method of alkalinephosphatase activity measurement is described in Example 2). As aresult, it was clarified that, for the maintenance of anundifferentiated state-maintaining ability, addition of not less than100 units of rLIF is preferable, and addition of not less than about1000 units or more, per 1 ml of culture medium is more preferable (FIG.3). From these results, an rLIF-supplemented culture medium was usedafter the inner cell mass formation.

(2) Consideration of Serum Addition

Conventionally, fetal bovine serum (FBS) is used for culturing ES cells.Whether or not to add FBS for rat ES cell establishment was examined.

To be specific, inner cell mass formation ability and formation ratewith or without (serum replacement reagent was used) addition of 20% FBSin the stage of forming inner cell mass from rat blastocyst wereexamined. In addition, maintenance or non-maintenance of theundifferentiated state-maintaining ability of ES cells at passage 1following primary culture was also examined. The maintenance ornon-maintenance of the undifferentiated state-maintaining ability wasjudged by the presence or absence of the alkaline phosphatase activityand embryoid body formation ability. As a result, when FBS was added,most of them were differentiated into multinuclear cells, withoutforming an inner cell mass (even if the mass is formed, it does notsufficiently grow to a size separable from trophoblast). The primarycultured ES cells were negative for the alkaline phosphatase activity,which is an index of undifferentiated state, and did not form a colonyor an embryoid body. In contrast, when a serum replacement reagent (KSR)was used instead of the serum, an inner cell mass capable of beingpicked up from the rat blastocyst was formed, the alkaline phosphataseactivity was also positive in the primary cultured ES cells, and acolony and an embryoid body were formed (FIG. 4). From the results, ithas been clarified that KSR is superior to FBS in rat ES cellestablishment. When the FBS concentration was 5% or 10%, the resultswere similar to those in the aforementioned 20% FBS.

(3) Composition of Culture Medium

Based on the examination results of the above-mentioned (1) and (2), aculture medium having the following composition was used forestablishing the rat ES cell.

Culture Medium for Rat ES Cell Establishment

It is a culture medium to be used in the stage of forming inner cellmass from blastocyst.

Dulbecco's modified Eagle medium/F12 (Asahi Techno Glass, Tokyo, Japan)380 ml

0.2M L-glutamine 5 ml

serum replacement reagent (KSR: Gibco BRL, Funakoshi, Tokyo, Japan) 100ml

non-essential amino acids (Gibco BRL, Funakoshi, Tokyo, Japan) 5 ml

Antibiotic-Antimicrotics solution (Gibco BRL, Funakoshi, Tokyo, Japan) 5ml

100 mM Na-pyruvate 5 ml

0.1M β mercaptoethanol 0.5 ml

Culture Medium for Rat ES Cell

It is a culture medium to be used for culture after formation of innercell mass.

Dulbecco's modified Eagle medium/F12 (Asahi Techno Glass, Tokyo, Japan)375 ml

0.2M L-glutamine 5 ml

serum replacement reagent (KSR: Gibco BRL, Funakoshi, Tokyo, Japan) 100ml

non-essential amino acids (Gibco BRL, Funakoshi, Tokyo, Japan) 5 ml

100-fold nucleoside stock solution 5 ml

(adenosine 4 mg, guanosine 4.25 mg, cytidine 3.65 mg, uridine 3.65 mg,thymidine 1.2 mg)

Antibiotic-Antimicrotics solution (Gibco BRL, Funakoshi, Tokyo, Japan) 5ml

100 mM Na-pyruvate 5 ml

0.1M β mercaptoethanol 0.5 ml

1000 U rat leukemia inhibitory factor

4) Consideration of Isolation Method of Rat ES Cell

The influence of trypsin treatment on isolation of the cell wasexamined. First of all, usual trypsin-EDTA treatment was performedduring the stage of isolating and sowing inner cell mass on a feedercell. As a result, most ES cells were differentiated. When cellaggregate was physically dissociated with a pipette, undifferentiatedstate could be maintained. It has been clarified that, also in thepassage of primary rat ES cell colony, when it is passaged with atrypsin-EDTA treatment, the cells are made to single cells anddifferentiate, while when it is passaged by dissociating a cell clampmechanically as mentioned above, the cells can maintain theirundifferentiated state.

Then, the influence of trypsin on the 2nd or later passages wasexamined. That is, difference in the maintenance of undifferentiatedstate was examined between, at the 2nd passage, when the cells arecompletely prepared to single cells using trypsin-EDTA and when thecells are passaged as aggregates consisting of 5-20 cells by mechanicaloperation at the end while minimizing treatment with trypsin-EDTA. As aresult, it has been clarified that passage in a single cell state causesspontaneous differentiation, whereas mechanical exfoliation of ES cellcolony and passage in an aggregate state consisting of 5-20 cells leadto maintenance of undifferentiated state (FIG. 5).

From the above results, it has been clarified that mouse ES cells aredesirably made completely to single cells with a trypsin-EDTA solution,and passaged, but rat ES cells are desirably passaged in an aggregatestate consisting of 5-20 cells, following mechanical exfoliation of EScell colony, because spontaneous differentiation occurs when the methodof mouse ES cell is applied to rat ES cells.

5) Establishment of Rat ES Cell

Based on the above examination results, rat ES cell was established. Theoutline of the establishment method is shown in FIG. 6.

After confirmation of rat blastocyst (late stage) under a microscope,zona pellucida was removed using Acidic Tyrode (pH 2.5). MitomycinC-treated normal mouse embryonic fibroblasts (feeder cells) were sown ona 60 mm gelatin-coated culture dish (Iwaki, Tokyo, Japan), the ratblastocysts (late stage) free of zona pellucida were transferred theretoby 5-10 blastocysts, and cultured in a culture medium for rat ES cellestablishment. The rat blastocyst (late stage) without zona pellucidaadhered to normal mouse embryonic fibroblasts at day 1-4, the inner cellmass (FIG. 7) alone was separated with a 200 μl pipette at day 5-10 fromthe adhesion. The culture medium for rat ES cell establishment (200 μl)was dispensed to a 1.5 ml sterile tube, the separated inner cell masswas transferred, and the cell aggregate was mechanically dissociatedwith a pipette. The dissociated inner cell mass was cultured in a mediumfor rat ES cell, in a 6-well gelatin-coated culture dish (Iwaki, Tokyo,Japan) containing feeder cells sown thereon the day before use. An EScell colony appeared in 2-4 days of culture (FIG. 8, and “primary EScell” in FIG. 6), and an ES cell colony (FIG. 9) at 5-10 days from theappearance of colony was separated with a 200 μl pipette whileconfirming the morphology under a microscope. In a 6-well gelatin-coatedculture dish (Iwaki, Tokyo, Japan) containing feeder cells sown thereonthe day before use, the mechanically dissociated ES cell colony wascultured in a culture medium for rat ES cell (in FIG. 6, “passage 1”).An ES cell colony appeared in 2-4 days of culture and passage becamepossible in 5-10 days.

Then, the culture medium was removed, the cells were washed twice withPBS(-) returned to room temperature, 2.5% trypsin (Gibco BRL, Funakoshi,Tokyo, Japan) incubated at 37° C. in advance was added at 500 μl per 60mm dish, and the solution was spread over the entire surface andimmediately removed. Exfoliation of not less than 70% of ES cellcolonies from the feeder cells was confirmed under a microscope and aculture medium (2 ml) containing 10% fetal bovine serum was added toquench the trypsin treatment. Using a 5 ml pipette, the ES cell colonieswere further mechanically exfoliated, and the cell suspension wascentrifuged (1000 rpm for 3 min, room temperature) to be separated intocells and culture medium, and the cells alone were recovered. The cellswere suspended in a culture medium for rat ES cell and, without completedissociation to single cells, formation of aggregates consisting of 5-20cells was confirmed under a microscope (FIG. 5, a)), and the cells werecultured in a 60 mm gelatin-coated culture dish (Iwaki, Tokyo, Japan)containing feeder cells sown thereon the day before use (in FIG. 6,“passage 2”). Thereafter, the cells stabilized to a passageable state atday 5-10 were passaged in the same manner as above and the cells atpassage 3 or later obtained were taken as established rat ES cells (FIG.10). The morphology of the established rat ES cell was typical to EScells and considerably similar to that of various ES cells heretoforereported (FIG. 11). The cells were passaged every 5-10 days in the samemanner as above even after the establishment and maintained.

Example 2 Analysis of Established Rat ES Cell

Using rat ES cells of passage 5, the presence of the property as ES cellwas confirmed. To be specific, each of the following properties wasexamined.

1) Analysis of Expression of Marker Gene for Undifferentiated State byRT-PCR Analysis

The presence or absence of expression of Oct3/4 and Nanog genes, whichare representative markers for undifferentiated state, in theestablished rat ES cells was confirmed. First of all, total RNA wasextracted from the established rat ES cells using ISOGEN (Nippon Gene,Tokyo, Japan). The single strand cDNA was synthesized in a mixture(total amount of 20 μl) including total RNA 2 μg, oligo(dT)₁₈ primers0.5 μl, dNTPs 10 pmol, RAV-2 RTase 5 units, and single strand synthesisbuffer (Takara, Kyoto, Japan). Synthesis was performed at 37° C. for 10min, 42° C. for 1 hr, 56° C. for 10 min, and 99° C. for 5 min Inaddition, the following primers were synthesized (the oligonucleotidesequences are described in the order of sense, antisense primers in theparenthesis, followed by annealing temperature, cycles for PCR, andlength of amplified fragment): β-actin (5′-AGAGCAAGAGAGGTATCCTG-3′ (SEQID NO:1), 5′-AGAGCATAGCCCTCGTAGAT-3′ (SEQ ID NO:2); 55° C.; 25 cycles;339 bp), Oct 3/4 (5′-ATGGACTACCCAGAACCCCAG-3′ (SEQ ID NO:3),5′-TTACAGGAGCTGCAGTTATAC-3′ (SEQ ID NO:4); 56° C.; 35 cycles; 448 bp),Nanog (5′-TAGCCCTGATTCTTCTAGCA-3′ (SEQ ID NO:5),(5′-TTTGCTGCAACGGCACATAA-3′ (SEQ ID NO:6); 54° C.; 35 cycles; 617 bp).The rat Oct 3/4 primers were designed by obtaining a CDS sequence ofmouse Oct 3/4 gene from GenBank, searching for its base sequence usingBLAST to obtain rat Oct 3/4 gene sequence, searching the homology ofmouse and rat, and selecting a region that is not amplified in the caseof mouse ES cell, which was used as rat specific Oct 3/4 primers.Primers for Nanog were designed based on the CDS sequences of mouse andhuman Nanog genes.

Amplification was performed in a mixture (total amount of 50 μl)including template cDNA 4 μl, 100 μM dNTPs, primers 10 pmol, Ex-Taq 1.0unit and Ex-Taq buffer (Takara, Kyoto, Japan). After PCR, a small amountof the mixture was electrophoresed on a 3.0% agarose gel, stained withethidium bromide (EtBr), and photographed under UV irradiation. Theresults are shown in FIG. 12. As is clear from FIG. 12, bands showingamplification of rat specific Oct3/4 and Nanog gene fragments wereconfirmed only in the cDNA of rat ES cell.

2) Analysis of Alkaline Phosphatase Activity

The presence or absence of alkaline phosphatase activity, which is oneof the representative indices verifying that the cell has anundifferentiated state-maintaining ability, was analyzed. First,established rat ES cells cultured in a culture medium for rat ES celland in the presence of feeder cell were directly fixed with 4%para-formaldehyde for 10 min, and thereafter fixed with 100% EtOH for 10min. The cells were washed with H₂O for 30 min. The alkaline phosphataseactivity was detected with Vector Red Alkaline Phosphatase Substrate KitI (Funakoshi, Tokyo, Japan) according to the manual. The results areshown in FIG. 13. Established rat ES cell was shown to be alkalinephosphatase activity positive.

3) Analysis of Embryoid Body Forming Ability

It is known that embryoid body is formed by culturing ES cells under theconditions free of feeder cell and LIF in a non-coated culture dish(Roy, S. et al., Mol. Cell. Biol., 18: 3947-3955 (1998)). Thus,established rat ES cells (1.0×10⁷ cells) were sown on an rLIF-freeculture medium for rat ES cell in a non-coated culture dish, andincubated at 37° C. for 20 days while exchanging the culture mediumevery 3 days. The results are shown in FIG. 14. As is clear from FIG.14, the embryoid body was formed, and many embryoid bodies showingcardiac muscle-like beating were confirmed.

4) ES Cell Marker Staining Analysis

The presence or absence of the expression of markers forundifferentiated state was examined. Established rat ES cells culturedin a culture medium for rat ES cell and in the presence of feeder cellwere directly fixed with 4% para-formaldehyde for 10 min and thereafterstood in 0.02% Triton X-100 for 15 min, and stood in 3% aqueous hydrogenperoxide/methanol for 15 min. After blocking with 5% serum/PBS for 15min, SSEA (stage-specific embryonic antigen)-1, SSEA-4, TRA-1-60 andTRA-1-81, each of which is a representative membrane protein of ES celland one kind of marker for undifferentiated state, were detected with EScell Characterization Kit (Funakoshi, Tokyo, Japan) and a secondaryantibody (anti-mouse IgG antibody-biotinylated (Funakoshi, Tokyo,Japan)), and a DAB staining kit (Kowa, Tokyo, Japan), according to themanual. The results are shown in FIG. 15. SSEA-1 and SSEA-4 werepositive and expression of these markers for undifferentiated state wasshown. As for TRA-1-60 and TRA-1-81, expression could not be detectedfrom the antibody staining results.

5) Analysis of Chromosome Number

Whether or not the established rat ES cell maintains the normalchromosome number was confirmed. The established rat ES cells werecultured in a culture medium for rat ES cell in a feeder cell-freegelatin-coated culture dish (Iwaki, Tokyo, Japan), and the chromosomenumber was analyzed by the G-banding method (Sumner, A. T. Cancer Genet.Cytogenet. 6: 59-87 (1982)). The results are shown in FIG. 16. Thenormal chromosome number (2n=42) of rat cells was shown to have beenmaintained.

6) Analysis of Number of Passages

The number of passages and undifferentiated state-maintaining abilitymaintenance rate of the established rat ES cell were examined. Rat EScells were cultured in a culture medium for rat ES cell in a feedercell-free gelatin-coated culture dish (Iwaki, Tokyo, Japan), and thenumber of passages and maintenance of undifferentiated state-maintainingability were examined with alkaline phosphatase activity as an index.The test was started with passage 5, and alkaline phosphatase activitystaining was conducted every 5 passages. The alkaline phosphataseactivity was detected using a Vector Red Alkaline Phosphatase SubstrateKit I (Funakoshi, Tokyo, Japan) according to the manual. The results areshown in FIG. 17. It has been clarified that the rat ES cells can becultured by the use of a culture medium for rat ES cell until at leastpassage 25, while maintaining a stable undifferentiatedstate-maintaining ability. It has been clarified that stable culture ispossible thereafter until passage 35.

7) Analysis of In Vitro Pluripotency

The spontaneous differentiation ability of the established rat ES cellwas examined. Established rat ES cells (1.0×10⁷ cells) were cultured ina culture medium for rat ES cell free of a rat leukemia inhibitoryfactor (rLIF) in a non-coated culture dish (Iwaki, Tokyo, Japan) toinitiate embryoid body formation. On day 7 from the start of theformation, the embryoid body was transferred to a gelatin-coated culturedish (Iwaki, Tokyo, Japan) and further cultured for 7 days. The resultsare shown in FIG. 18.

As shown in FIG. 18(a), neuron-like cells emerged from the embryoidbody. In the enlarged view (b) of the neuron-like cells, an imagewherein neurite is extended between two cells to connect them isconfirmed. While the adipose-like cell is characterized in that itcontains many clear granules in the cytosol, as shown in FIG. 18(c), anadipose-like cell image containing many clear granules was confirmed. Asshown in FIG. 18(d), many epithelium-lineage cells were also confirmed.As evidenced, it has been confirmed that the established rat ES cell hasan ability to spontaneously differentiate to various cells.

8) Influences of Serum and LIF Addition on Rat ES Cell

Influences of LIF and serum on the established rat ES cell in themaintenance of the undifferentiated state-maintaining ability and growthability were examined. Rat ES cell (passage 5) were subcultured for onegeneration under the following 4 kinds of culture conditions. (1)containing rat LIF (rLIF), serum-free (containing 20% KSR), (2) withoutLIF, serum-free (containing 20% KSR), (3) containing mouse LIF (mLIF),serum-free (containing 20% KSR), and (4) containing rat LIF (rLIF),containing 20% serum.

Whether or not the undifferentiated state-maintaining ability wasmaintained was examined by analyzing the presence or absence ofexpression of each gene of Oct3/4, Nanog and Rex-1, which areundifferentiated state-marker genes of ES cell. In addition, thepresence or absence of expression of ERas gene, which is important forteratoma formation characteristic of ES cells was also analyzed. Thesequence of the primers used, annealing temperature and cycles of PCRare as shown below.

Oct 3/4: (SEQ ID NO: 3) 5′-ATGGACTACCCAGAACCCCAG-3′, (SEQ ID NO: 4)5′-TTACAGGAGCTGCAGTTATAC-3′, 56° C., 40 cycles, Nanog: (SEQ ID NO: 5)5′-TAGCCCTGATTCTTCTAGCA-3′, (SEQ ID NO: 6) 5′-TTTGCTGCAACGGCACATAA-3′60° C., 40 cycles, Rex-1: (SEQ ID NO: 7) 5′-AAATCATGACGAGGCAAGGC-3′(SEQ ID NO: 8) 5′-TGAGTTCGCTCCAACAGTCT-3′ 60° C., 40 cycles, ERas:(SEQ ID NO: 9) 5′-ACCTGAGCCCCGGCACACAG-3′ (SEQ ID NO: 10)5′-CAGCTGCAGCGGTGTGGGCG-3′ 64° C., 40 cycles.

Furthermore, a morphological observation was also performed. The resultsare shown in FIG. 19.

When rat ES cells were cultured using rLIF and 20% KSR (theaforementioned (1)), they were morphologically stable (FIG. 19), andexpression of the ES cell marker genes was observed. Thus, it has beenconfirmed that cell growth is possible while maintaining theundifferentiated state-maintaining ability. On the other hand, whencultivated under the other 3 conditions (the aforementioned (2)-(4)),expression of characteristic ES cell markers disappeared, and a shift toa morphologically differentiated state was observed (FIG. 19). From theabove results, it has been clarified that that both rat LIF and serumreplacement reagent (KSR) are important for the culture of rat ES cells.

9) Analysis of Teratoma Forming Ability

Established rat ES cells are adjusted to 1.0×10⁷ cells/200 μl with PBSto give a suspension, and subjected to cell transplantation to theinside of the testis and the subdermic of syngeneic male rat(15-week-old). The rat is sacrificed, and the formed teratoma is excisedand fixed with 4% para-formaldehyde. A tissue section is prepared,stained with hematoxylin/eosin, and differentiation induction to threeembryonic germs (endoderm, mesoderm and ectoderm) is confirmed under amicroscope.

10) Analysis of Chimeric Rat-Producing Ability

A chicken albumin promoter is incorporated into a pEGFP-1 vector(clonetech) at the upstream of EGFP gene and constructed as a vectorpermitting stable intracellular expression of EGFP gene. The preparedvector was introduced into the rat ES cell by the electroporationmethod, a cell line (RESC/EGFP strain), in which the vector has beenincorporated into the chromosomal gene in the rat ES cell wasestablished by selection using a drug G418.

Separately, an ES cell-syngeneic rat was naturally crossbred and, on day3 after confirmation of vaginal plug, the female rat for oocyte samplingwas sacrificed. The uterus was excised, perfused with MW medium andoocytes were recovered. Thereafter, 40 sampled oocytes are developed ina 5% CO₂ incubator up to 8-cell embryo. Zona pellucida is removed in anoil drop using Acidic Tyrode (pH 2.5), the feeder cell is removed, onecell aggregate (5 to 20 cells) of the rat ES cell line (RESC/EGFP cells)established above is introduced into the fertilized oocyte of thesyngeneic rat by the microinjection method. The oocyte fortransplantation is cultured overnight in a 5% CO₂ culture apparatus.Separately, a previously vasoligated male rat and a female rat foroocyte sampling are crossed, vaginal plug of the female rat is confirmedthe next day to give a pseudopregnant female rat. The anesthetizedpseudopregnant female rat is opened, and 10 oocytes for transplantationare placed in the uterus. The offspring is removed by natural childbirthor caesarotomy when particularly needed, the offspring rat is exposed tofluorescence irradiation and the birth of a chimeric rat is confirmed bythe fluorescent body color.

Example 3 Establishment of ES Cell and Analysis of Established Rat ESCell (2) 1) Establishment of ES Cell

In the same manner as in the case of Wistar Kyoto rat (WKY) shown inExample 1, ES cell was also established in Wistar Hannover GALAS rat(WHG) and Brown Norway rat (BN). As a result, ES cells of both ratscould be established. The photographs of the inner cell masses formedfrom the blastocysts of respective rats (WKY, WHG and BN) are shown inFIG. 20. The photographs of the established ES cells (passage 5) arealso shown in FIG. 20. It was confirmed that, in both the WHG rat EScell and BN rat ES cell, subculture while holding the undifferentiatedstate-maintaining ability as in WKY rat ES cell was possible.

2) Analysis of Expression of ES Cell Marker Genes by RT-PCR Analysis

The presence or absence of the expression of ES cell marker genes in theestablished WKY rat ES cell, WHG rat ES cell and BN rat ES cell wasconfirmed by RT-PCR analysis. The experiment was performed in the samemanner as in Example 2, 1). Expression of Oct3/4, Rex-1 and Nanog genes,which are undifferentiated state-marker genes of ES cell was analyzed.Expression of ERas gene important for teratoma formation characteristicof ES cell was also analyzed. As the primers for RT-PCR, those shown inExample 2, 1) and 8) were used. The results of WKY rat ES cell are shownin FIG. 21. Expression was observed for all factors of Oct3/4, Rex-1,Nanog and ERas. The same results were obtained for WHG rat ES cell andBN rat ES cell.

3) Analysis of Embryoid Body Formed from ES Cell

Established rat ES cells were sown in a culture medium for rat ES cellfree of rLIF in a non-coated culture dish, and the cells were incubatedat 37° C. for 20 days, during which the culture medium was exchangedevery 3 days. As a result, an embryoid body was formed, and manyembryoid bodies showing beating like cardiac muscle were confirmed.

Then, RNA was extracted from the embryoid body having cardiomyocyte-likecells, and expression of each marker gene of neuron (ectoderm),cardiomyocyte (mesoderm) and hepatocyte (endoderm) was analyzed byRT-PCR. The name of each marker gene, primer sequences, annealingtemperature and cycles of PCR are as shown below.

A) neuron marker Nestin: (SEQ ID NO: 11) 5′-GCTCTGACCTATCATCTGAG-3′,(SEQ ID NO: 12) 5′-AGATGCACAGGAGATGCTAC-3′, 58° C., 40 cycles, TUJI:(SEQ ID NO: 13) 5′-GGAACGCATCAGTGTCTACT-3′, (SEQ ID NO: 14)5′-ACCACGCTGAAGGTGTTCAT-3′ 60° C., 40 cycles, MSI-1: (SEQ ID NO: 15)5′-TCTCACTGCTTATGGTCCGA-3′, (SEQ ID NO: 16)5′-TCAGTGGTACCCATTGGTGA-3′, 60° C., 40 cycles, GFAP: (SEQ ID NO: 17)5′-GGCTCTGAGAGAGATTCGCA-3′, (SEQ ID NO: 18)5′-ATGTCCAGGGCTAGCTTAAC-3′, 58° C., 40 cycles, B) cardiomyocyte markerCCC: (SEQ ID NO: 19) 5′-TCTGAAGCGGCAGAAGAATC-3′ (SEQ ID NO: 20)5′-TGACCTCGATGAACTTGGGA-3′ 58° C., 40 cycles, ANF: (SEQ ID NO: 21)5′-ATACAGTGCGGTGTCCAACA-3′ (SEQ ID NO: 22) 5′-TTATCTTCGGTACCGGAAGC-3′58° C., 40 cycles, KvLQT1: (SEQ ID NO: 23) 5′-TGCGGATGCTGCATGTTGAT-3′(SEQ ID NO: 24) 5′-CAAACCCAGAGCCAAGTATG-3′, 58° C., 40 cycles,C) hepatocyte marker ALB: (SEQ ID NO: 25) 5′-GCTTGCTGTGATAAGCCAGT-3′,(SEQ ID NO: 26) 5′-TGGCAGACAGATAGTCTTCC-3′ 58° C., 40 cycles, TDO:(SEQ ID NO: 27) 5′-CGATGAGAAGCGTCATGACT-3′, (SEQ ID NO: 28)5′-AACCAGGTACGATGAGAGGT-3′, 58° C., 40 cycles, TAT: (SEQ ID NO: 29)5′-AATGAGATTCGAGACGGGCT-3′, (SEQ ID NO: 30)5′-TTCATCACAGTGGTAGTGCT-3′, 58° C., 40 cycles, G6P: (SEQ ID NO: 31)5′-GTCAACGTATGGATTCCGGT-3′, (SEQ ID NO: 32) 5′-GTTCTCCTTTGCAGCTCTTG-3′58° C., 40 cycles.

The results of RT-PCR are shown in FIG. 22. It has been clarified thatgenes free of expression in an undifferentiated state (lane 1) allturned positive in the embryonic body (lane 2). From the results, it hasbeen clarified that established rat ES cells have an ability todifferentiate to the cells of three embryonic germ (ectoderm, mesoderm,endoderm) lineages, namely, they have the properties of ES cell.

4) Analysis of Teratoma Formation Ability

The established WKY rat ES cell, WHG rat ES cell, and BN rat ES cell(2.5×10⁷ cells) were each subcutaneously transplanted to immunodeficientmice. One or two months later, formation of teratoma was confirmed inall of these ES cell-transplanted mice. The results of WHG rat ES cellare shown in FIG. 23. The mice were sacrificed, formed teratoma wasexcised and fixed with 4% para-formaldehyde. Tissue sections wereprepared and stained with hematoxylin/eosin (FIG. 23). By observationunder a microscope, respective tissues of glandular structure (mesodermlineage), blood vessel endotheloid structure (ectoderm lineage),intestine-like structure (endoderm lineage), and osteocyte-likestructure (mesoderm lineage) were observed. From the above results, ithas been clarified that the established rat ES cells have ateratoma-forming ability, and the formed teratomas have three embryonicgerm (endoderm, mesoderm and ectoderm) structures.

5) Analysis of Chimeric Rat-Producing Ability

pCAG-EGFP gene was introduced into a rat ES cell (WHG rat ES cell) bythe electroporation method to prepare a WHG rat ES cell line(hereinafter to be abbreviated as pCAG-EGFP/WHGrES cell line) in whichpCAG-EGFP gene is integrated into a chromosome.

A female rat syngeneic with WHG rat ES cell was naturally crossbred and,after confirmation of the presence of vaginal plug the next day, oocyteswere sampled on day 4. Thereafter, fertilized oocytes developed to theblastocyst stage in a 5% CO₂ incubator were harvested, and 8-12pCAG-EGFP/WHGr ES cells were transplanted per 1 oocyte by themicroinjection method (hereinafter to be abbreviated as ES(+)).

Separately, a vasoligated syngeneic male rat and a syngeneic female ratwere crossed and the presence of vaginal plug was confirmed the nextday, to prepare a pseudopregnant female rat. On day 4 of confirmation ofthe presence of vaginal plug, 6-10 ES(+)oocytes were transplanted per 1pseudopregnant rat. Thereafter, the offspring rat (GFP chimeric rat) wasexcised by caesarotomy.

Various tissues were separated from the GFP chimeric rat, and genomicDNA was extracted. Using the genomic DNA as a template and GFP specificprimers, genomic PCR was performed according to a conventional method.The results are shown in FIG. 24. As a result, a band corresponding toGFP gene suggesting pCAG-EGFP/WHGrES cell derivation was confirmed inall organs examined except 3 organs (thymus, blood vessel, spleen). Incontrast, a band corresponding to GFP gene was not confirmed in thecontrol (wild-type rat) genome. It has been clarified that theestablished rat ES cell has a chimeric rat-producing ability, since, asshown above, a gene derived from rat ES cell (pCAG-EGFP/WHGrES cell) wascontained in each tissue or cell.

6) Analysis of Chimeric Rat Tissue

Using the GFP chimeric rat obtained in the aforementioned 5) and asyngeneic wild-type rat, each tissue was sectioned, and subjected toimmunostaining using a GFP antibody (CLONTECH). The results are shown inFIG. 25. DAB-staining was confirmed only in the tissues of GFP chimericrat, and it has been clarified that ES cell derived GFP-positive cellwas certainly present in each tissue and expressed GFP.

The liver section of the GFP chimeric rat was fluorescence stained withHoechst (blue) and rhodamin (red). As a result, a red region showingGFP-positive cells was confirmed near the central vein (data not shown).

From the above results, it has been clarified that not only a rat EScell-derived gene is contained in each tissue but also the ES celldifferentiated to the cell of each tissue lineage by morphologicalobservation.

INDUSTRIAL APPLICABILITY

The present invention provides a rat ES cell. The established rat EScell of the present invention has first enabled preparation ofgenetically modified rats (knockout rat, knockin rat etc.), and can bewidely used for pharmacological studies and physiological studies invarious regions such as cancer and cranial nerve, and further, studieson regenerative medicine and the like.

Sequence Listing Free Text

The base sequence described in SEQ ID NO:1 is a PCR primer.

The base sequence described in SEQ ID NO:2 is a PCR primer.

The base sequence described in SEQ ID NO:3 is a PCR primer.

The base sequence described in SEQ ID NO:4 is a PCR primer.

The base sequence described in SEQ ID NO:5 is a PCR primer.

The base sequence described in SEQ ID NO:6 is a PCR primer.

The base sequence described in SEQ ID NO:7 is a PCR primer.

The base sequence described in SEQ ID NO:8 is a PCR primer.

The base sequence described in SEQ ID NO:9 is a PCR primer.

The base sequence described in SEQ ID NO:10 is a PCR primer.

The base sequence described in SEQ ID NO:11 is a PCR primer.

The base sequence described in SEQ ID NO:12 is a PCR primer.

The base sequence described in SEQ ID NO:13 is a PCR primer.

The base sequence described in SEQ ID NO:14 is a PCR primer.

The base sequence described in SEQ ID NO:15 is a PCR primer.

The base sequence described in SEQ ID NO:16 is a PCR primer.

The base sequence described in SEQ ID NO:17 is a PCR primer.

The base sequence described in SEQ ID NO:18 is a PCR primer.

The base sequence described in SEQ ID NO:19 is a PCR primer.

The base sequence described in SEQ ID NO:20 is a PCR primer.

The base sequence described in SEQ ID NO:21 is a PCR primer.

The base sequence described in SEQ ID NO:22 is a PCR primer.

The base sequence described in SEQ ID NO:23 is a PCR primer.

The base sequence described in SEQ ID NO:24 is a PCR primer.

The base sequence described in SEQ ID NO:25 is a PCR primer.

The base sequence described in SEQ ID NO:26 is a PCR primer.

The base sequence described in SEQ ID NO:27 is a PCR primer.

The base sequence described in SEQ ID NO:28 is a PCR primer.

The base sequence described in SEQ ID NO:29 is a PCR primer.

The base sequence described in SEQ ID NO:30 is a PCR primer.

The base sequence described in SEQ ID NO:31 is a PCR primer.

The base sequence described in SEQ ID NO:32 is a PCR primer.

1. A heterozygous knockout or konckin rat obtained by (I) producing achimeric rat by a process comprising the following steps (X)-(Z): (X) astep for introducing a targeting vector into a target gene in the genomeof a rat embryonic stem cell by homologous recombination to produce ahomologously recombined rat embryonic stem cell, (Y) a step forpreparing a chimeric rat embryo for transplantation by (i) injecting therat embryonic stem cell into which the gene was introduced into ablastocoele of a rat blastocyst or a morula stage-, 16-cell stage- or8-cell stage-rat embryo or (ii) co-culturing the rat embryonic stem cellinto which the gene was introduced with an 8-cell stage- or 16-cellstage-rat embryo without zona pellucidas, (Z) a step for transferringthe chimeric rat embryo for transplantation into the uterus of apseudopregnant female rat to produce the chimeric rat, wherein the ratembryonic stem cell has the following properties (a)-(k): (a) expressingOct3/4 gene and Nanog gene, (b) positive for alkaline phosphataseactivity, (c) having an embryoid body forming ability, (d) expressingSSEA (Stage-Specific Embryonic Antigen)-1 and SSEA-4, (e) having thesame number of chromosomes as does a normal rat cell, (f) capable ofbeing subcultured and holding the undifferentiated state, (g) having invitro pluripotency, (h) having a potential to differentiate for cells ofthree embryonic germ lineages, (i) having teratoma formation ability,(j) having an ability to produce a chimeric rat, (k) beinggermline-competent, wherein the rat embryonic stem cell is obtained bythe method consisting essentially of the following steps (A)-(E)performed using a culture medium with 2% or less serum concentration:(A) culturing a rat blastocyst on inactivated mouse embryonicfibroblasts in a leukemia inhibitory factor (LIF)-free culture medium toform an inner cell mass in the blastocyst, (B) dissociating the innercell mass, wherein the dissociated inner cell mass is in a cellaggregate state, (C) culturing primary embryonic stem cells resultingfrom a culture of the dissociated inner cell mass on inactivated mouseembryonic fibroblasts until the primary embryonic stem cells can bepassaged, (D) dissociating the primary embryonic stem cells, which canbe passaged, wherein the dissociated primary embryonic stem cells are ina cell aggregate state, (E) culturing the dissociated primary embryonicstem cells on inactivated mouse embryonic fibroblasts to establish anembryonic stem cell, wherein an rLIF-containing culture medium is usedin steps (C)-(E); and (II) crossing the chimeric rat with a wildtyperat, thereby producing a heterozygous knockout or knockin rat.
 2. Ahomologous knockout or knockin rat obtained by mating the heterozygousknockout or knockin rats according to claim 1.